Expression of the stem cell factor in fibroblasts, endothelial cells, and macrophages in periapical tissues in human chronic periapical diseases.

Science.gov (United States)

Shen, S Q; Wang, R; Huang, S G

2017-03-08

Stem cell factor (SCF), an important stem cell cytokine, has multiple functions. Fibroblasts (FBs), mature mast cells, endothelial cells (ECs), and eosinophil granulocytes can produce SCF in the inflammatory process. Therefore, we aimed to observe SCF expression in FBs, ECs, and macrophages (MPs) in periapical tissues in human chronic periapical disease and investigate the effects of cells expressing SCF in pathogenesis of the disease. Healthy (N = 20), periapical cyst (N = 15), and periapical granuloma (N = 15) tissues were fixed in 10% formalin for 48 h, embedded in paraffin, and stained with hematoxylin and eosin to observe histological changes. SCF expression was observed in FBs, ECs, and MPs in periapical tissues by double immunofluorescence. CD334, CD31, and CD14 are specific markers of FBs, ECs, and MPs, respectively. Results showed that densities of CD334-SCF double-positive FBs, CD31-SCF double-positive ECs, and CD14-SCF double-positive MPs were significantly increased in periapical tissue groups (P periapical tissue groups (P > 0.05). CD14-SCF double-positive MP density was considerably higher in periapical granulomas than in cysts (P periapical tissues, suggesting that the cells might be related to occurrence, development, and pathogenesis of chronic periapical disease.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

Science.gov (United States)

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

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Paggetti, Jerome; Haderk, Franziska; Seiffert, Martina; Janji, Bassam; Distler, Ute; Ammerlaan, Wim; Kim, Yeoun Jin; Adam, Julien; Lichter, Peter; Solary, Eric; Berchem, Guy

2015-01-01

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin–positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. PMID:26100252

The impact of vascular endothelial growth factor and basic fibroblast growth factor on cardiac fibroblasts grown under altered gravity conditions

DEFF Research Database (Denmark)

Ulbrich, Claudia; Leder, Annekatrin; Pietsch, Jessica

2010-01-01

Myocardium is very sensitive to gravitational changes. During a spaceflight cardiovascular atrophy paired with rhythm problems and orthostatic intolerance can occur. The aim of this study was to investigate the impact of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor...

CXCL10 can inhibit endothelial cell proliferation independently of CXCR3.

Directory of Open Access Journals (Sweden)

Gabriele S V Campanella

2010-09-01

Full Text Available CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10 is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

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Alves, Tercia Rodrigues [Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Laboratorio de Biologia da Celula Endotelial e da Angiogenese (LabAngio), Rio de Janeiro (Brazil); Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Carvalho da Fonseca, Anna Carolina [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Nunes, Sara Santana; Oliveira da Silva, Aline [Universidade do Estado do Rio de Janeiro (UERJ), Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biologia Celular, Laboratorio de Biologia da Celula Endotelial e da Angiogenese (LabAngio), Rio de Janeiro (Brazil); Dubois, Luiz Gustavo Feijo; Faria, Jane; Kahn, Suzana Assad [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); Viana, Nathan Bessa [Universidade Federal do Rio de Janeiro, Laboratorio de Pincas Oticas, Coordenacao de Programas de Estudos Avancados, Instituto de Ciencias Biomedicas, Rio de Janeiro (Brazil); Universidade Federal do Rio de Janeiro, Instituto de Fisica, Rio de Janeiro (Brazil); Marcondes, Jorge [Universidade Federal do Rio de Janeiro, Hospital Universitario Clementino Fraga Filho, Servico de Neurocirurgia, Rio de Janeiro (Brazil); Legrand, Chantal [Institut Universitaire d' Hematologie, Universite Paris-Diderot, Paris 7, INSERM U553, Paris (France); Moura-Neto, Vivaldo [Universidade Federal do Rio de Janeiro (UFRJ), Programa de Biologia Celular e do Desenvolvimento, Instituto de Ciencias Biomedicas, INNT/INCT/MCT, Rio de Janeiro (Brazil); and others

2011-09-10

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Tenascin-C in the extracellular matrix promotes the selection of highly proliferative and tubulogenesis-defective endothelial cells

International Nuclear Information System (INIS)

Alves, Tercia Rodrigues; Carvalho da Fonseca, Anna Carolina; Nunes, Sara Santana; Oliveira da Silva, Aline; Dubois, Luiz Gustavo Feijo; Faria, Jane; Kahn, Suzana Assad; Viana, Nathan Bessa; Marcondes, Jorge; Legrand, Chantal; Moura-Neto, Vivaldo

2011-01-01

The extracellular matrix (ECM) contains important cues for tissue homeostasis and morphogenesis. The matricellular protein tenascin-C (TN-C) is overexpressed in remodeling tissues and cancer. In the present work, we studied the effect of different ECM-which exhibited a significant diversity in their TN-C content-in endothelial survival, proliferation and tubulogenic differentiation: autologous (endothelial) ECM devoid of TN-C, but bearing large amounts of FN; fibroblast ECM, bearing both high TN-C and FN contents; and finally, glioma-derived matrices, usually poor in FN, but very rich in TN-C. HUVECs initially adhered to the immobilized matrix produced by U373 MG glioma cells, but significantly detached and died by anoikis (50 to 80%) after 24 h, as compared with cells incubated with endothelial and fibroblast matrices. Surviving endothelial cells (20 to 50%) became up to 6-fold more proliferative and formed 74-97% less tube-like structures in vitro than cells grown on non-tumoral matrices. An antibody against the EGF-like repeats of tenascin-C (TN-C) partially rescued cells from the tubulogenic defect, indicating that this molecule is responsible for the selection of highly proliferative and tubulogenic defective endothelial cells. Interestingly, by using defined substrata, in conditions that mimic glioma and normal cell ECM composition, we observed that fibronectin (FN) modulates the TN-C-induced selection of endothelial cells. Our data show that TN-C is able to modulate endothelial branching morphogenesis in vitro and, since it is prevalent in matrices of injured and tumor tissues, also suggest a role for this protein in vascular morphogenesis, in these physiological contexts.

Vascular endothelial growth factor and basic fibroblast growth factor expression positively correlates with angiogenesis and peritumoural brain oedema in astrocytoma

International Nuclear Information System (INIS)

Jang, F.F.; Wei, W.

2008-01-01

Astrocytoma is the most malignant intracranial neoplasm and is characterized by high neovascularization and peritumoural brain oedema. Angiogenesis is a complicated process in oncogenesis regulated by the balance between angiogenic and antiangiogenic factors. The expression of two angiogenic growth factors, vascular endothelial growth factor and basic fibroblast growth factor were investigated using immunohistochemistry for astrocytoma from 82 patients and 11 normal human tissues. The expression of vascular endothelial growth factor and basic fibroblast growth factor positively correlate with the pathological grade of astrocytoma, microvessel density numbers and brain oedema, which may be responsible for the increased tumour neovascularization and peritumoural brain oedema. The results support the idea that inhibiting vascular endothelial growth factor and basic fibroblast growth factor are useful for the treatment of human astrocytoma and to improve patient's clinical outcomes and prognosis. (author)

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

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Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

DEFF Research Database (Denmark)

Barnathan, E S; Kuo, A; Karikó, K

1990-01-01

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence...

Pathology of experimental Ebola virus infection in African green monkeys. Involvement of fibroblastic reticular cells.

Science.gov (United States)

Davis, K J; Anderson, A O; Geisbert, T W; Steele, K E; Geisbert, J B; Vogel, P; Connolly, B M; Huggins, J W; Jahrling, P B; Jaax, N K

1997-08-01

Ebola virus has been responsible for explosive lethal outbreaks of hemorrhagic fever in both humans and nonhuman primates. Previous studies showed a predilection of Ebola virus for cells of the mononuclear phagocyte system and endothelial cells. To examine the distribution of lesions and Ebola virus antigen in the tissues of six adult male African green monkeys (Cercopithecus aethiops) that died 6 to 7 days after intraperitoneal inoculation of Ebola-Zaire (Mayinga) virus. Tissues were examined histologically, immunohistochemically, and ultrastructurally. A major novel finding of this study was that fibroblastic reticular cells were immunohistochemically and ultrastructurally identified as targets of Ebola virus infection. The role of Ebola virus-infected fibroblastic reticular cells in the pathogenesis of Ebola hemorrhagic fever warrants further investigation. This is especially important because of recent observations indicating that fibroblastic reticular cells, along with the reticular fibers they produce, maximize the efficiency of the immune response.

Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

International Nuclear Information System (INIS)

Laranjeira, Marta S; Carvalho, Ângela; Ferraz, Maria Pia; Monteiro, Fernando Jorge; Pelaez-Vargas, Alejandro; Hansford, Derek; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena

2014-01-01

Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior. (paper)

Fibrosis in connective tissue disease: the role of the myofibroblast and fibroblast-epithelial cell interactions

Science.gov (United States)

Krieg, Thomas; Abraham, David; Lafyatis, Robert

2007-01-01

Fibrosis, characterized by excessive extracellular matrix accumulation, is a common feature of many connective tissue diseases, notably scleroderma (systemic sclerosis). Experimental studies suggest that a complex network of intercellular interactions involving endothelial cells, epithelial cells, fibroblasts and immune cells, using an array of molecular mediators, drives the pathogenic events that lead to fibrosis. Transforming growth factor-β and endothelin-1, which are part of a cytokine hierarchy with connective tissue growth factor, are key mediators of fibrogenesis and are primarily responsible for the differentiation of fibroblasts toward a myofibroblast phenotype. The tight skin mouse (Tsk-1) model of cutaneous fibrosis suggests that numerous other genes may also be important. PMID:17767742

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

Science.gov (United States)

Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

2018-01-01

Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

Isolated tumor endothelial cells maintain specific character during long-term culture

International Nuclear Information System (INIS)

Matsuda, Kohei; Ohga, Noritaka; Hida, Yasuhiro; Muraki, Chikara; Tsuchiya, Kunihiko; Kurosu, Takuro; Akino, Tomoshige; Shih, Shou-Ching

2010-01-01

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1-2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

[The role of endothelial cells and endothelial precursor cells in angiogenesis].

Science.gov (United States)

Poreba, Małgorzata; Usnarska-Zubkiewicz, Lidia; Kuliczkowski, Kazimierz

2006-01-01

Endothelium plays a key role in maintenance of vascular homeostasis in human organism. According to new data endothelial cells and hematopoietic cells have a common precursor in prenatal life--a hemangioblast, which explains the fact of sharing the same determinants on the surface of both type of cells. Circulating endothelial precursors were identified in adults and this suggests that hemangioblasts may be present not only during embriogenesis. In some clinical situations the increased numbers of endothelial cells and endothelial precursors were noted, and especially in patients with neoplastic diseases, which is probably the result of increased angiogenesis. Endothelial precursors are thought to be the promice for therapeutic purposes in future--to increase local angiogenesis.

Ets2 in tumor fibroblasts promotes angiogenesis in breast cancer.

Directory of Open Access Journals (Sweden)

Julie A Wallace

Full Text Available Tumor fibroblasts are active partners in tumor progression, but the genes and pathways that mediate this collaboration are ill-defined. Previous work demonstrates that Ets2 function in stromal cells significantly contributes to breast tumor progression. Conditional mouse models were used to study the function of Ets2 in both mammary stromal fibroblasts and epithelial cells. Conditional inactivation of Ets2 in stromal fibroblasts in PyMT and ErbB2 driven tumors significantly reduced tumor growth, however deletion of Ets2 in epithelial cells in the PyMT model had no significant effect. Analysis of gene expression in fibroblasts revealed a tumor- and Ets2-dependent gene signature that was enriched in genes important for ECM remodeling, cell migration, and angiogenesis in both PyMT and ErbB2 driven-tumors. Consistent with these results, PyMT and ErbB2 tumors lacking Ets2 in fibroblasts had fewer functional blood vessels, and Ets2 in fibroblasts elicited changes in gene expression in tumor endothelial cells consistent with this phenotype. An in vivo angiogenesis assay revealed the ability of Ets2 in fibroblasts to promote blood vessel formation in the absence of tumor cells. Importantly, the Ets2-dependent gene expression signatures from both mouse models were able to distinguish human breast tumor stroma from normal stroma, and correlated with patient outcomes in two whole tumor breast cancer data sets. The data reveals a key function for Ets2 in tumor fibroblasts in signaling to endothelial cells to promote tumor angiogenesis. The results highlight the collaborative networks that orchestrate communication between stromal cells and tumor cells, and suggest that targeting tumor fibroblasts may be an effective strategy for developing novel anti-angiogenic therapies.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea

Science.gov (United States)

Lopes, Flavia C. M.; Traina, Fabiola; Almeida, Camila B.; Leonardo, Flavia C.; Franco-Penteado, Carla F.; Garrido, Vanessa T.; Colella, Marina P.; Soares, Raquel; Olalla-Saad, Sara T.; Costa, Fernando F.; Conran, Nicola

2015-01-01

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the

Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma.

Science.gov (United States)

Le, Tran; New, Jacob; Jones, Joel W; Usman, Shireen; Yalamanchali, Sreeya; Tawfik, Ossama; Hoover, Larry; Bruegger, Dan E; Thomas, Sufi Mary

2017-10-01

Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation. After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups. Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039). AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA. © 2017 ARS

Post-transcriptional regulation of osteoblastic platelet-derived growth factor receptor-alpha expression by co-cultured primary endothelial cells

DEFF Research Database (Denmark)

Finkenzeller, Günter; Mehlhorn, Alexander T; Schmal, Hagen

2010-01-01

-alpha downregulation is dependent on time and cell number. This effect was specific to endothelial cells and was not observed when hOBs were co-cultured with human primary chondrocytes or fibroblasts. Likewise, HUVEC-mediated suppression of PDGFR-alpha expression was only seen in hOBs and mesenchymal stem cells......Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in osteoblast function. Inhibition of PDGFR activity leads to a suppression of osteoblast proliferation, whereas mineralized matrix production is enhanced. In previous experiments, we showed that co......-cultivation of human primary endothelial cells and human primary osteoblasts (hOBs) leads to a cell contact-dependent downregulation of PDGFR-alpha expression in the osteoblasts. In this study, we investigated this effect in more detail, revealing that human umbilical vein endothelial cell (HUVEC)-mediated PDGFR...

Endothelial-regenerating cells: an expanding universe.

Science.gov (United States)

Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

2010-03-01

Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

International Nuclear Information System (INIS)

Zhang Yueqing; Hu Xiaobo; Tian Ruiyang; Wei Wangui; Hu Wei; Chen Xia; Han Wei; Chen Huayou; Gong Yi

2006-01-01

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the α v -containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism

The hematopoietic chemokine CXCL12 promotes integration of human endothelial colony forming cell-derived cells into immature vessel networks.

Science.gov (United States)

Newey, Sarah E; Tsaknakis, Grigorios; Khoo, Cheen P; Athanassopoulos, Thanassi; Camicia, Rosalba; Zhang, Youyi; Grabowska, Rita; Harris, Adrian L; Roubelakis, Maria G; Watt, Suzanne M

2014-11-15

Proangiogenic factors, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF-2) prime endothelial cells to respond to "hematopoietic" chemokines and cytokines by inducing/upregulating expression of the respective chemokine/cytokine receptors. Coculture of human endothelial colony forming cell (ECFC)-derived cells with human stromal cells in the presence of VEGF and FGF-2 for 14 days resulted in upregulation of the "hematopoietic" chemokine CXCL12 and its CXCR4 receptor by day 3 of coculture. Chronic exposure to the CXCR4 antagonist AMD3100 in this vasculo/angiogenesis assay significantly reduced vascular tubule formation, an observation recapitulated by delayed AMD3100 addition. While AMD3100 did not affect ECFC-derived cell proliferation, it did demonstrate a dual action. First, over the later stages of the 14-day cocultures, AMD3100 delayed tubule organization into maturing vessel networks, resulting in enhanced endothelial cell retraction and loss of complexity as defined by live cell imaging. Second, at earlier stages of cocultures, we observed that AMD3100 significantly inhibited the integration of exogenous ECFC-derived cells into established, but immature, vascular networks. Comparative proteome profiler array analyses of ECFC-derived cells treated with AMD3100 identified changes in expression of potential candidate molecules involved in adhesion and/or migration. Blocking antibodies to CD31, but not CD146 or CD166, reduced the ECFC-derived cell integration into these extant vascular networks. Thus, CXCL12 plays a key role not only in endothelial cell sensing and guidance, but also in promoting the integration of ECFC-derived cells into developing vascular networks.

Wound Healing Activity of Extracts and Formulations of Aloe vera, Henna, Adiantum capillus-veneris, and Myrrh on Mouse Dermal Fibroblast Cells.

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Negahdari, Samira; Galehdari, Hamid; Kesmati, Mahnaz; Rezaie, Anahita; Shariati, Gholamreza

2017-01-01

Among the most important factors in wound healing pathways are transforming growth factor beta1 and vascular endothelial growth factor. Fibroblasts are the main cell in all phases wound closure. In this study, the extracts of plant materials such as Adiantum capillus-veneris , Commiphora molmol , Aloe vera , and henna and one mixture of them were used to treatment of normal mouse skin fibroblasts. Cytotoxic effects of each extract and their mixture were assessed on mouse skin fibroblasts cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We performed migration assays to assess migration properties of mouse skin fibroblasts cells in response to the extracts. Changes in the gene expression of the Tgf β1 and Vegf-A genes were monitored by real-time polymerase chain reaction. A. capillus-veneris , C. molmol and henna extract improved the expression of Tgfβ1 gene. All used extracts upregulated the expression of Vegf-A gene and promoted the migration of mouse fibroblast cells in vitro . The present study demonstrated that the mentioned herbal extracts might be effective in wound healing, through the improvement in the migration of fibroblast cells and regulating the gene expression of Tgfβ1 and Vegf-A genes in fibroblast cells treated with extracts.

Biofabrication enables efficient interrogation and optimization of sequential culture of endothelial cells, fibroblasts and cardiomyocytes for formation of vascular cords in cardiac tissue engineering

International Nuclear Information System (INIS)

Iyer, Rohin K; Radisic, Milica; Chiu, Loraine L Y; Vunjak-Novakovic, Gordana

2012-01-01

We previously reported that preculture of fibroblasts (FBs) and endothelial cells (ECs) prior to cardiomyocytes (CMs) improved the structural and functional properties of engineered cardiac tissue compared to culture of CMs alone or co-culture of all three cell types. However, these approaches did not result in formation of capillary-like cords, which are precursors to vascularization in vivo. Here we hypothesized that seeding the ECs first on Matrigel and then FBs 24 h later to stabilize the endothelial network (sequential preculture) would enhance cord formation in engineered cardiac organoids. Three sequential preculture groups were tested by seeding ECs (D4T line) at 8%, 15% and 31% of the total cell number on Matrigel-coated microchannels and incubating for 24 h. Cardiac FBs were then seeded (32%, 25% and 9% of the total cell number, respectively) and incubated an additional 24 h. Finally, neonatal rat CMs (60% of the total cell number) were added and the organoids were cultivated for seven days. Within 24 h, the 8% EC group formed elongated cords which eventually developed into beating cylindrical organoids, while the 15% and 31% EC groups proliferated into flat EC monolayers with poor viability. Excitation threshold (ET) in the 8% EC group (3.4 ± 1.2 V cm −1 ) was comparable to that of the CM group (3.3 ± 1.4 V cm −1 ). The ET worsened with increasing EC seeding density (15% EC: 4.4 ± 1.5 V cm −1 ; 31% EC: 4.9 ± 1.5 V cm −1 ). Thus, sequential preculture promoted vascular cord formation and enhanced architecture and function of engineered heart tissues. (paper)

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

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Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

2013-05-03

Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial

An Expandable, Inducible Hemangioblast State Regulated by Fibroblast Growth Factor

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David T. Vereide

2014-12-01

Full Text Available During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that “trap” murine cells in a proliferative state and endow them with a hemangioblast potential. These “expandable” hemangioblasts (eHBs are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines.

Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

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Ryohei Numata

Full Text Available The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

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Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

2016-07-01

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

α-Klotho expression determines nitric oxide synthesis in response to FGF-23 in human aortic endothelial cells.

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Chih-Ping Chung

Full Text Available Endothelial cells (ECs express fibroblast growth factor (FGF receptors and are metabolically active after treatment with FGF-23. It is not known if this effect is α-Klotho independent or mediated by humoral or endogenous endothelial α-Klotho. In the present study, we aimed to characterize EC α-Klotho expression within the human vascular tree and to investigate the potential role of α-Klotho in determining FGF-23 mediated EC regulation. Human tissue and ECs from various organs were used for immunohistochemistry and Western blot. Primary cultures of human aortic endothelial cells (HAECs and human brain microvascular endothelial cells (HBMECs were used to generate in vitro cell models. We found endogenous α-Klotho expression in ECs from various organs except in microvascular ECs from human brain. Furthermore, FGF-23 stimulated endothelial nitric oxide synthase (eNOS expression, nitric oxide (NO production, and cell proliferation in HAECs. Interestingly, these effects were not observed in our HBMEC model in vitro. High phosphate treatment and endothelial α-Klotho knockdown mitigated FGF-23 mediated eNOS induction, NO production, and cell proliferation in HAECs. Rescue treatment with soluble α-Klotho did not reverse endothelial FGF-23 resistance caused by reduced or absent α-Klotho expression in HAECs. These novel observations provide evidence for differential α-Klotho functional expression in the human endothelium and its presence may play a role in determining the response to FGF-23 in the vascular tree. α-Klotho was not detected in cerebral microvascular ECs and its absence may render these cells nonresponsive to FGF-23.

Analysis of the receptor-mediated B19V mechanism of internalization in the endothelial B19V infection and adenovirus-mediated reactivation of B19V in endothelial cells

OpenAIRE

Pozzuto, Tanja

2012-01-01

Human Parvovirus B19 (B19V) is the causative agent of erythema infectiosum, hydrops fetalis, aplastic crises and polyarthritis. B19V displays a very narrow cell and tissue tropism with productive infection thought to be restricted exclusively to erythroid progenitor cells in the bone marrow and fetal liver. However, over the last years increasing evidence for the presence of B19V DNA in other cell types and tissues such as synovial fibroblasts, tonsilles and skin as well as endothelial cells ...

Synergistic inhibition of endothelial cell proliferation, tube formation, and sprouting by cyclosporin A and itraconazole.

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Benjamin A Nacev

Full Text Available Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA, an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC(50 dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.

Synergistic inhibition of endothelial cell proliferation, tube formation, and sprouting by cyclosporin A and itraconazole.

Science.gov (United States)

Nacev, Benjamin A; Liu, Jun O

2011-01-01

Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC(50) dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.

TGF-β2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation

International Nuclear Information System (INIS)

Lu Jiawei; Lu Zhenyu; Reinach, Peter

2006-01-01

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-β2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-β2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-β2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-β2 and FGF-2 oppositely affect BCE cell proliferation and TGF-β2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-β2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-β2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-β2-induced suppression of the PI3-kinase/AKT signaling pathway

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

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Suellen D S Oliveira

Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

Signaling hierarchy regulating human endothelial cell development.

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Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

Protective role of microRNA-29a in denatured dermis and skin fibroblast cells after thermal injury

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Jie Zhou

2016-03-01

Full Text Available Our previous study has suggested that downregulated microRNA (miR-29a in denatured dermis might be involved in burn wound healing. However, the exact role of miR-29a in healing of burn injury still remains unclear. Here, we found that expression of miR-29a was notably upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury, and thereafter gradually downregulated compared with control group. By contrast, the expression of collagen, type I, alpha 2 (COL1A2 and vascular endothelial growth factor (VEGF-A were first reduced and subsequently upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury. We further identified COL1A2 as a novel target of miR-29a, which is involved in type I collagen synthesis, and showed that miR-29a negatively regulated the expression level of COL1A2 in skin fibroblast cells. In addition, VEGF-A, another target gene of miR-29a, was also negatively mediated by miR-29a in skin fibroblast cells. Inhibition of miR-29a expression significantly promoted the proliferation and migration of skin fibroblast cells after thermal injury, and knockdown of COL1A2 and VEGF-A reversed the effects of miR-29a on the proliferation and migration of skin fibroblast cells. Furthermore, we found that Notch2/Jagged2 signaling was involved in miR-29a response to burn wound healing. Our findings suggest that downregulated miR-29a in denatured dermis may help burn wound healing in the later phase, probably via upregulation of COL1A2 and VEGF-A expression, which can further enhance type I collagen synthesis and angiogenesis.

Reduced Ang2 expression in aging endothelial cells

International Nuclear Information System (INIS)

Hohensinner, P.J.; Ebenbauer, B.; Kaun, C.; Maurer, G.; Huber, K.; Wojta, J.

2016-01-01

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

Reduced Ang2 expression in aging endothelial cells

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Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

Mesenchymal stem cells induce dermal fibroblast responses to injury

International Nuclear Information System (INIS)

Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

2010-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

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Shumei Man

2008-01-01

Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties.

Science.gov (United States)

Kubota, Hiroshi; Wu, Xin; Goodyear, Shaun M; Avarbock, Mary R; Brinster, Ralph L

2011-08-01

Previous studies suggest that exogenous factors crucial for spermatogonial stem cell (SSC) self-renewal are conserved among several mammalian species. Since glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are critical for rodent SSC self-renewal, we hypothesized that they might promote self-renewal of nonrodent SSCs. Therefore, we cultured testicular germ cells from prepubertal rabbits in the presence of GDNF and FGF2 and found they proliferated indefinitely as cellular clumps that displayed characteristics previously identified for rodent SSCs. The rabbit germ cells could not be maintained on mouse embryonic fibroblast (STO) feeders that support rodent SSC self-renewal in vitro but were rather supported on mouse yolk sac-derived endothelial cell (C166) feeder layers. Proliferation of rabbit germ cells was dependent on GDNF. Of critical importance was that clump-forming rabbit germ cells colonized seminiferous tubules of immunodeficient mice, proliferated for at least 6 mo, while retaining an SSC phenotype in the testes of recipient mice, indicating that they were rabbit SSCs. This study demonstrates that GDNF is a mitogenic factor promoting self-renewal that is conserved between rodent and rabbit SSCs; with an evolutionary separation of ∼ 60 million years. These findings provide a foundation to study the mechanisms governing SSC self-renewal in nonrodent species.

Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

Science.gov (United States)

Ollivier, V; Bentolila, S; Chabbat, J; Hakim, J; de Prost, D

1998-04-15

The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts.

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Cieslik, Katarzyna A; Trial, JoAnn; Crawford, Jeffrey R; Taffet, George E; Entman, Mark L

2014-05-01

Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-β), due to reduced expression of TGF-β receptor I (TβRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-β promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-β signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TβRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ". © 2013. Published by Elsevier Ltd. All rights reserved.

Urokinase receptor expression on human microvascular endothelial cells is increased by hypoxia: Implications for capillary-like tube formation in a fibrin matrix

NARCIS (Netherlands)

Kroon, M.E.; Koolwijk, P.; Vecht, B. van der; Hinsbergh, V.W.M. van

2000-01-01

Hypoxia stimulates angiogenesis, the formation of new blood vessels. This study evaluates the direct effect of hypoxia (1% oxygen) on the angiogenic response of human microvascular endothelial cells (hMVECs) seeded on top of a 3-dimensional fibrin matrix, hMVECs stimulated with fibroblast growth

Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

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Zhang, J.C. [Department of Vascular Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Zheng, G.F. [Department of Vascular Surgery, The People' s Hospital of Ganzhou, Ganzhou (China); Wu, L.; Ou Yang, L.Y.; Li, W.X. [Department of Vascular Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

2014-08-08

Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs) expressing human basic fibroblast growth factor (hbFGF). After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC), MSCs expressing hbFGF (hbFGF-MSC), MSC controls, and phosphate-buffered saline (PBS) controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF) expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001); however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008) and microvessel density (P<0.001). Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats

Directory of Open Access Journals (Sweden)

J.C. Zhang

2014-10-01

Full Text Available Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs expressing human basic fibroblast growth factor (hbFGF. After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC, MSCs expressing hbFGF (hbFGF-MSC, MSC controls, and phosphate-buffered saline (PBS controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001; however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008 and microvessel density (P<0.001. Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.

Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

Science.gov (United States)

Mohammadi, Elham; Nassiri, Seyed Mahdi; Rahbarghazi, Reza; Siavashi, Vahid; Araghi, Atefeh

2015-12-01

Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

Chromosome aberration induction in human diploid fibroblast and epithelial cells

International Nuclear Information System (INIS)

Scott, D.

1986-01-01

The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

Endothelial cells and hematopoiesis: a light microscopic study of fetal, normal, and pathologic human bone marrow in plastic-embedded sections.

Science.gov (United States)

Islam, A; Glomski, C; Henderson, E S

1992-07-01

The origin and morphological identity of hematopoietic progenitor cells, as well as their precursor, the pleuripotential hematopoietic stem cell (HSC), has not been established. Our studies of 2 microns sectioned undecalcified plastic-embedded bone marrow (BM) from healthy human fetuses; normal adults; patients with acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic granulocytic leukemia (CGL) in various stages (chronic, accelerated, acute blastic phase, and after autografting); and patients recovering from therapy-induced marrow hypoplasia suggest that proliferative hematopoietic zones exist near the endosteum (endosteal marrow) and the vascular endothelium (capillary and sinus-lining endothelium) and a maturational zone distal to these regions. In some of these areas, morphologically recognizable hematopoietic cells were seen and interpreted as emerging and maturing in a sequential progression, suggesting an origin from the endosteal or endothelial progenitors. In other loci, early hematopoietic cells were seen in close contact with the endosteal or vascular endothelial (VE) cells. This latter relationship suggested that these areas of cellular contact were important and represented sites of cell to cell interaction that may be associated with the liberation of growth factors by endosteal and endothelial cells and their action on hematopoietic progenitor cells. Following treatment-induced hypoplasia, the endosteal and VE cells were seen to modulate, transform, and migrate into the surrounding empty and edematous marrow space as fibroblasts. Later, as hemopoietic regeneration began, clusters of regenerating hematopoietic cells were seen adjacent to bone trabecule (BT) and near the vascular endothelium. We postulate that endosteal and VE cells are the equivalent of embryonal-stage, undifferentiated mesenchyme and, under the appropriate regulatory influence, are capable of modulation and transformation (differentiation) into stromal

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

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Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

2011-08-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

Science.gov (United States)

Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

2011-01-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

Human endothelial precursor cells express tumor endothelial marker 1/endosialin/CD248.

Science.gov (United States)

Bagley, Rebecca G; Rouleau, Cecile; St Martin, Thia; Boutin, Paula; Weber, William; Ruzek, Melanie; Honma, Nakayuki; Nacht, Mariana; Shankara, Srinivas; Kataoka, Shiro; Ishida, Isao; Roberts, Bruce L; Teicher, Beverly A

2008-08-01

Angiogenesis occurs during normal physiologic processes as well as under pathologic conditions such as tumor growth. Serial analysis of gene expression profiling revealed genes [tumor endothelial markers (TEM)] that are overexpressed in tumor endothelial cells compared with normal adult endothelial cells. Because blood vessel development of malignant tumors under certain conditions may include endothelial precursor cells (EPC) recruited from bone marrow, we investigated TEM expression in EPC. The expression of TEM1 or endosialin (CD248) and other TEM has been discovered in a population of vascular endothelial growth factor receptor 2+/CD31+/CD45-/VE-cadherin+ EPC derived from human CD133+/CD34+ cells. EPC share some properties with fully differentiated endothelial cells from normal tissue, yet reverse transcription-PCR and flow cytometry reveal that EPC express higher levels of endosialin at the molecular and protein levels. The elevated expression of endosialin in EPC versus mature endothelial cells suggests that endosialin is involved in the earlier stages of tumor angiogenesis. Anti-endosialin antibodies inhibited EPC migration and tube formation in vitro. In vivo, immunohistochemistry indicated that human EPC continued to express endosialin protein in a Matrigel plug angiogenesis assay established in nude mice. Anti-endosialin antibodies delivered systemically at 25 mg/kg were also able to inhibit circulating murine EPC in nude mice bearing s.c. SKNAS tumors. EPC and bone marrow-derived cells have been shown previously to incorporate into malignant blood vessels in some instances, yet they remain controversial in the field. The data presented here on endothelial genes that are up-regulated in tumor vasculature and in EPC support the hypothesis that the angiogenesis process in cancer can involve EPC.

Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

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Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

2017-09-15

Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

Endothelial cell repopulation after stenting determines in-stent neointima formation: effects of bare-metal vs. drug-eluting stents and genetic endothelial cell modification.

Science.gov (United States)

Douglas, Gillian; Van Kampen, Erik; Hale, Ashley B; McNeill, Eileen; Patel, Jyoti; Crabtree, Mark J; Ali, Ziad; Hoerr, Robert A; Alp, Nicholas J; Channon, Keith M

2013-11-01

Understanding endothelial cell repopulation post-stenting and how this modulates in-stent restenosis is critical to improving arterial healing post-stenting. We used a novel murine stent model to investigate endothelial cell repopulation post-stenting, comparing the response of drug-eluting stents with a primary genetic modification to improve endothelial cell function. Endothelial cell repopulation was assessed en face in stented arteries in ApoE(-/-) mice with endothelial-specific LacZ expression. Stent deployment resulted in near-complete denudation of endothelium, but was followed by endothelial cell repopulation, by cells originating from both bone marrow-derived endothelial progenitor cells and from the adjacent vasculature. Paclitaxel-eluting stents reduced neointima formation (0.423 ± 0.065 vs. 0.240 ± 0.040 mm(2), P = 0.038), but decreased endothelial cell repopulation (238 ± 17 vs. 154 ± 22 nuclei/mm(2), P = 0.018), despite complete strut coverage. To test the effects of selectively improving endothelial cell function, we used transgenic mice with endothelial-specific overexpression of GTP-cyclohydrolase 1 (GCH-Tg) as a model of enhanced endothelial cell function and increased NO production. GCH-Tg ApoE(-/-) mice had less neointima formation compared with ApoE(-/-) littermates (0.52 ± 0.08 vs. 0.26 ± 0.09 mm(2), P = 0.039). In contrast to paclitaxel-eluting stents, reduced neointima formation in GCH-Tg mice was accompanied by increased endothelial cell coverage (156 ± 17 vs. 209 ± 23 nuclei/mm(2), P = 0.043). Drug-eluting stents reduce not only neointima formation but also endothelial cell repopulation, independent of strut coverage. In contrast, selective targeting of endothelial cell function is sufficient to improve endothelial cell repopulation and reduce neointima formation. Targeting endothelial cell function is a rational therapeutic strategy to improve vascular healing and decrease neointima formation after stenting.

Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

International Nuclear Information System (INIS)

Basso, F G; Pansani, T N; Turrioni, A P S; Hebling, J; De Souza Costa, C A; Kurachi, C; Bagnato, V S

2013-01-01

Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm −2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal–Wallis and Mann–Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions. (paper)

Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

Science.gov (United States)

Basso, F. G.; Pansani, T. N.; Turrioni, A. P. S.; Kurachi, C.; Bagnato, V. S.; Hebling, J.; de Souza Costa, C. A.

2013-05-01

Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm-2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions.

Sickle erythrocytes inhibit human endothelial cell DNA synthesis

International Nuclear Information System (INIS)

Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K.

1990-01-01

Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

Science.gov (United States)

Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

Directory of Open Access Journals (Sweden)

Annie O Smith

Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

Science.gov (United States)

Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

2017-01-01

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

Science.gov (United States)

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Cell proliferation in vitro modulates fibroblast collagenase activity

International Nuclear Information System (INIS)

Lindblad, W.J.; Flood, L.

1986-01-01

Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

Reduced Ang2 expression in aging endothelial cells.

Science.gov (United States)

Hohensinner, P J; Ebenbauer, B; Kaun, C; Maurer, G; Huber, K; Wojta, J

2016-06-03

Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. Copyright © 2016 Elsevier Inc. All rights reserved.

Strategies to reverse endothelial progenitor cell dysfunction in diabetes.

Science.gov (United States)

Petrelli, Alessandra; Di Fenza, Raffaele; Carvello, Michele; Gatti, Francesca; Secchi, Antonio; Fiorina, Paolo

2012-01-01

Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial), their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs). Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Strategies to Reverse Endothelial Progenitor Cell Dysfunction in Diabetes

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Alessandra Petrelli

2012-01-01

Full Text Available Bone-marrow-derived cells-mediated postnatal vasculogenesis has been reported as the main responsible for the regulation of vascular homeostasis in adults. Since their discovery, endothelial progenitor cells have been depicted as mediators of postnatal vasculogenesis for their peculiar phenotype (partially staminal and partially endothelial, their ability to differentiate in endothelial cell line and to be incorporated into the vessels wall during ischemia/damage. Diabetes mellitus, a condition characterized by cardiovascular disease, nephropathy, and micro- and macroangiopathy, showed a dysfunction of endothelial progenitor cells. Herein, we review the mechanisms involved in diabetes-related dysfunction of endothelial progenitor cells, highlighting how hyperglycemia affects the different steps of endothelial progenitor cells lifetime (i.e., bone marrow mobilization, trafficking into the bloodstream, differentiation in endothelial cells, and homing in damaged tissues/organs. Finally, we review preclinical and clinical strategies that aim to revert diabetes-induced dysfunction of endothelial progenitor cells as a means of finding new strategies to prevent diabetic complications.

Isolation of a cDNA for a Growth Factor of Vascular Endothelial Cells from Human Lung Cancer Cells: Its Identity with Insulin‐like Growth Factor II

Science.gov (United States)

Hagiwara, Koichi; Kobayashi, Tatsuo; Tobita, Masato; Kikyo, Nobuaki; Yazaki, Yoshio

1995-01-01

We have found growth‐promoting activity for vascular endothelial cells in the conditioned medium of a human lung cancer cell line, T3M‐11. Purification and characterization of the growth‐promoting activity have been carried out using ammonium sulfate precipitation and gel‐exclusion chromatography. The activity migrated as a single peak just after ribonuclease. It did not bind to a heparin affinity column. These results suggest that the activity is not a heparin‐binding growth factor (including fibroblast growth factors) or a vascular endothelial growth factor. To identify the molecule exhibiting the growth‐promoting activity, a cDNA encoding the growth factor was isolated through functional expression cloning in COS‐1 cells from a cDNA library prepared from T3M‐11 cells. The nucleotide sequence encoded by the cDNA proved to be identical with that of insulin‐like growth factor II. PMID:7730145

Endothelial induced EMT in breast epithelial cells with stem cell properties

DEFF Research Database (Denmark)

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

2011-01-01

endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D......492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close...

Endothelial cells in the eyes of an immunologist.

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Young, M Rita

2012-10-01

Endothelial cell activation in the process of tumor angiogenesis and in various aspects of vascular biology has been extensively studied. However, endothelial cells also function in other capacities, including in immune regulation. Compared to the more traditional immune regulatory populations (Th1, Th2, Treg, etc.), endothelial cells have received far less credit as being immune regulators. Their regulatory capacity is multifaceted. They are critical in both limiting and facilitating the trafficking of various immune cell populations, including T cells and dendritic cells, out of the vasculature and into tissue. They also can be induced to stimulate immune reactivity or to be immune inhibitory. In each of these parameters (trafficking, immune stimulation and immune inhibition), their role can be physiological, whereby they have an active role in maintaining health. Alternatively, their role can be pathological, whereby they contribute to disease. In theory, endothelial cells are in an ideal location to recruit cells that can mediate immune reactivity to tumor tissue. Furthermore, they can activate the immune cells as they transmigrate across the endothelium into the tumor. However, what is seen is the absence of these protective effects of endothelial cells and, instead, the endothelial cells succumb to the defense mechanisms of the tumor, resulting in their acquisition of a tumor-protective role. To understand the immune regulatory potential of endothelial cells in protecting the host versus the tumor, it is useful to better understand the other circumstances in which endothelial cells modulate immune reactivities. Which of the multitude of immune regulatory roles that endothelial cells can take on seems to rely on the type of stimulus that they are encountering. It also depends on the extent to which they can be manipulated by potential dangers to succumb and contribute toward attack on the host. This review will explore the physiological and pathological roles

Metabolic cooperation between co-cultured lung cancer cells and lung fibroblasts.

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Koukourakis, Michael I; Kalamida, Dimitra; Mitrakas, Achilleas G; Liousia, Maria; Pouliliou, Stamatia; Sivridis, Efthimios; Giatromanolaki, Alexandra

2017-11-01

Cooperation of cancer cells with stromal cells, such as cancer-associated fibroblasts (CAFs), has been revealed as a mechanism sustaining cancer cell survival and growth. In the current study, we focus on the metabolic interactions of MRC5 lung fibroblasts with lung cancer cells (A549 and H1299) using co-culture experiments and studying changes of the metabolic protein expression profile and of their growth and migration abilities. Using western blotting, confocal microscopy and RT-PCR, we observed that in co-cultures MRC5 respond by upregulating pyruvate dehydrogenase (PDH) and the monocarboxylate transporter MCT1. In contrast, cancer cells increase the expression of glucose transporters (GLUT1), LDH5, PDH kinase and the levels of phosphorylated/inactivated pPDH. H1299 cells growing in the same culture medium with fibroblasts exhibit a 'metastasis-like' phenomenon by forming nests within the fibroblast area. LDH5 and pPDH were drastically upregulated in these nests. The growth rate of both MRC5 and cancer cells increased in co-cultures. Suppression of LDHA or PDK1 in cancer cells abrogates the stimulatory signal from cancer cells to fibroblasts. Incubation of MRC5 fibroblasts with lactate resulted in an increase of LDHB and of PDH expression. Silencing of PDH gene in fibroblasts, or silencing of PDK1 or LDHA gene in tumor cells, impedes cancer cell's migration ability. Overall, a metabolic cooperation between lung cancer cells and fibroblasts has been confirmed in the context of direct Warburg effect, thus the fibroblasts reinforce aerobic metabolism to support the intensified anaerobic glycolytic pathways exploited by cancer cells.

Atlantic salmon endothelial cells from the heart were more susceptible than fibroblasts from the bulbus arteriosus to four RNA viruses but protected from two viruses by dsRNA pretreatment.

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Pham, Phuc H; Tong, Winnie W L; Misk, Ehab; Jones, Ginny; Lumsden, John S; Bols, Niels C

2017-11-01

Heart diseases caused by viruses are major causes of Atlantic salmon aquaculture loss. Two Atlantic salmon cardiovascular cell lines, an endothelial cell line (ASHe) from the heart and a fibroblast cell line (BAASf) from the bulbus arteriosus, were evaluated for their response to four fish viruses, CSV, IPNV, VHSV IVa and VHSV IVb, and the innate immune agonist, double-stranded RNA mimic poly IC. All four viruses caused cytopathic effects in ASHe and BAASf. However, ASHe was more susceptible to all four viruses than BAASf. When comparing between the viruses, ASHe cells were found to be moderately susceptible to CSV and VHSV IVb, but highly susceptible to IPNV and VHSV IVa induced cell death. All four viruses were capable of propagating in the ASHe cell line, leading to increases in virus titre over time. In BAASf, CSV and IPNV produced more than one log increase in titre from initial infection, but VHSV IVb and IVa did not. When looking at the antiviral response of both cell lines, Mx proteins were induced in ASHe and BAASf by poly IC. All four viruses induced Mx proteins in BAASf, while only CSV and VHSV IVb induced Mx proteins in ASHe. IPNV and VHSV IVa suppressed Mx proteins expression in ASHe. Pretreatment of ASHe with poly IC to allow for Mx proteins accumulation protected the culture from subsequent infections with IPNV and VHSV IVa, resulting in delayed cell death, reduced virus titres and reduced viral proteins expression. These data suggest that endothelial cells potentially can serve as points of infections for viruses in the heart and that two of the four viruses, IPNV and VHSV IVa, have mechanisms to avoid or downregulate antiviral responses in ASHe cells. Furthermore, the high susceptibility of the ASHe cell line to IPNV and VHSV IVa can make it a useful tool for studying antiviral compounds against these viruses and for general detection of fish viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Magnetizable stent-grafts enable endothelial cell capture

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Tefft, Brandon J. [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Uthamaraj, Susheil [Division of Engineering, Mayo Clinic, Rochester, MN (United States); Harburn, J. Jonathan [School of Medicine, Pharmacy and Health, Durham University, Stockton-on-Tees (United Kingdom); Hlinomaz, Ota [Department of Cardioangiology, St. Anne' s University Hospital, Brno (Czech Republic); Lerman, Amir [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States); Dragomir-Daescu, Dan [Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN (United States); Sandhu, Gurpreet S., E-mail: sandhu.gurpreet@mayo.edu [Department of Cardiovascular Diseases, Mayo Clinic, Rochester, MN (United States)

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance. - Highlights: • Magnetic stent-grafts were made from 2205 steel stents and polyurethane nanofibers. • Stent-grafts remained patent and formed a thin and uniform neointima when implanted. • Stent-grafts captured endothelial cells labeled with magnetic nanoparticles. �

Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

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David M Harris

Full Text Available Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza and the growth factors (GF granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

Nipah virus infection and glycoprotein targeting in endothelial cells

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Maisner Andrea

2010-11-01

Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

Radioprotection of mouse CNS endothelial cells in vivo

International Nuclear Information System (INIS)

Lyubimova, N.; Coultas, P.; Martin, R.

1996-01-01

Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy γ-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

Adhesion and endothelialization of endothelial cells on the surface of endovascular stents by the novel rotational culture of cells

International Nuclear Information System (INIS)

Tang Chaojun; Wang Guixue; Cao Yi; Wu Xue; Xie Xiang; Xiao Li

2008-01-01

Recent researches indicate that the initial event in the implantation of endovascular stents involves mechanical injury to the vessel wall. Confluent endothelialization of vascular grafts in vitro before implantation has been suggested as a way to reduce injury of the blood vessel. The purpose of this study is to establish a useful way to improve the adhesion of endothelial cells and accelerate endothelialization on the surface of endovascular stents by a novel rotational culture device. Numerical simulation was used to predict the shear stress on the surface of stents. The number of cellular adhesion was calculated by cell counting, the cell growth was observed by scanning electron microscope and fluorescence microscope. Numerical simulation results showed that the stents was exposed to shear stress of 2.66 x 10 -3 to 8.88 x 10 -2 Pa. Rotational culture of human umbilical vein endothelial cells could enhance the adhesion of cells and accelerate endothelialization on the surface of stents when the culture conditions for EC adhesion were intermediate rotation speed, higher dynamic incubation times, lower cell densities

Differentiation state determines neural effects on microvascular endothelial cells

International Nuclear Information System (INIS)

Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

2012-01-01

Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. ► Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. ► Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. ► Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell production of nitric oxide. ► Neural progenitor cells and dorsal root

Tumor and Endothelial Cell Hybrids Participate in Glioblastoma Vasculature

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Soufiane El Hallani

2014-01-01

Full Text Available Background. Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM. Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM. Materials and Methods. We used EGFR FISH Probe to detect EGFR amplification and anti-CD31, CD105, VE-cadherin, and vWF to identify endothelial cells. Endothelial and GBM cells were grown separately, labeled with GFP and DsRed lentiviruses, and then cocultured with or without contact. Results. In a subset of GBM tissues, we found that several tumor endothelial cells carry EGFR amplification, characteristic of GBM tumor cells. This observation was reproduced in vitro: when tumor stem cells derived from GBM were grown in the presence of human endothelial cells, a fraction of them acquired endothelial markers (CD31, CD105, VE-cadherin, and vWF. By transduction with GFP and DsRed expressing lentiviral vectors, we demonstrate that this phenomenon is due to cell fusion and not transdifferentiation. Conclusion. A fraction of GBM stem cells thus has the capacity to fuse with endothelial cells and the resulting hybrids may participate in tumor microvascular proliferation and in treatment resistance.

Syncytin is involved in breast cancer-endothelial cell fusions

DEFF Research Database (Denmark)

Bjerregaard, Bolette; Holck, S.; Christensen, I.J.

2006-01-01

Cancer cells can fuse spontaneously with normal host cells, including endothelial cells, and such fusions may strongly modulate the biological behaviour of tumors. However, the underlying mechanisms are unknown. We now show that human breast cancer cell lines and 63 out of 165 (38%) breast cancer...... specimens express syncytin, an endogenous retroviral envelope protein, previously implicated in fusions between placental trophoblast cells. Additionally, endothelial and cancer cells are shown to express ASCT-2, a receptor for syncytin. Syncytin antisense treatment decreases syncytin expression...... and inhibits fusions between breast cancer cells and endothelial cells. Moreover, a syncytin inhibitory peptide also inhibits fusions between cancer and endothelial cells. These results are the first to show that syncytin is expressed by human cancer cells and is involved in cancer-endothelial cell fusions....

Endothelial induced EMT in breast epithelial cells with stem cell properties.

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Valgardur Sigurdsson

Full Text Available Epithelial to mesenchymal transition (EMT is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad to N-Cadherin (N-Cad and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high/CD24(low ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Endothelial induced EMT in breast epithelial cells with stem cell properties.

Science.gov (United States)

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J R; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A; Petersen, Ole William; Magnusson, Magnus K; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression of keratins, a switch from E-Cadherin (E-Cad) to N-Cadherin (N-Cad) and enhanced migration. Acquisition of cancer stem cell associated characteristics like increased CD44(high)/CD24(low) ratio, resistance to apoptosis and anchorage independent growth was also seen in D492M cells. Endothelial induced EMT in D492 was partially blocked by inhibition of HGF signaling. Basal-like breast cancer, a vascular rich cancer with stem cell properties and adverse prognosis has been linked with EMT. We immunostained several basal-like breast cancer samples for endothelial and EMT markers. Cancer cells close to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal-like breast cancer contains cells with an EMT phenotype, most prominently close to vascular rich areas of these tumors. We conclude that endothelial cells are potent inducers of EMT and may play a role in progression of basal-like breast cancer.

Oxidative stress induced pulmonary endothelial cell proliferation is ...

African Journals Online (AJOL)

Cellular hyper-proliferation, endothelial dysfunction and oxidative stress are hallmarks of the pathobiology of pulmonary hypertension. Indeed, pulmonary endothelial cells proliferation is susceptible to redox state modulation. Some studies suggest that superoxide stimulates endothelial cell proliferation while others have ...

Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

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Hou, Jingang; Kim, Sunchang

2018-05-05

Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 μM H 2 O 2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-β1) and beta 2 (TGF-β2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

Effects of external radiation in a co-culture model of endothelial cells and adipose-derived stem cells

International Nuclear Information System (INIS)

Haubner, Frank; Leyh, Michaela; Ohmann, Elisabeth; Pohl, Fabian; Prantl, Lukas; Gassner, Holger G

2013-01-01

The inflammatory response clinically observed after radiation has been described to correlate with elevated expression of cytokines and adhesion molecules by endothelial cells. Therapeutic compensation for this microvascular compromise could be an important approach in the treatment of irradiated wounds. Clinical reports describe the potential of adipose-derived stem cells to enhance wound healing, but the underlying cellular mechanisms remain largely unclear. Human dermal microvascular endothelial cells (HDMEC) and human adipose-derived stem cells (ASC) were cultured in a co-culture setting and irradiated with sequential doses of 2 to 12 Gy. Cell count was determined 48 h after radiation using a semi-automated cell counting system. Levels of interleukin-6 (IL-6), basic fibroblast growth factor (FGF), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were determined in the supernatants using enzyme-linked immunosorbent assay (ELISA). Irradiated HDMEC and ASC as well as non-irradiated co-cultures, HDMEC or ASC respectively were used as controls. Cell count was significantly reduced in irradiated co-cultures of HDMEC and ASC compared to non-irradiated controls. Levels of IL-6, FGF, ICAM-1 and VCAM-1 in the supernatants of the co-cultures were significantly less affected by external radiation in comparison to HDMEC. The increased expression of cytokines and adhesion molecules by HDMEC after external radiation is mitigated in the co-culture setting with ASC. These in vitro changes seem to support the clinical observation that ASC may have a stabilizing effect when injected into irradiated wounds

Comparison of the phosphorylation events in membranes prepared from proliferating versus quiescent endothelial cells

International Nuclear Information System (INIS)

Kazlauskas, A.; DiColeto, P.E.

1986-01-01

Little is known of the intracellular events which regulate the proliferation of endothelial cells (EC). Triton-solubilized membranes from proliferating (sparse) and quiescent (confluent) EC were incubated at pH 6.5 in the presence of divalent cations and [ 32 P]ATP. Membrane proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The overall kinase activity per mg protein was slightly greater in membranes prepared from proliferating versus quiescent cells. They found four proteins labeled in sparse cells to a dramatically greater extent having the following approximate molecular masses: 180, 100, 97 and 55 kilodalton (kd). The first two phosphoproteins were phosphorylated on serine residues exclusively; the 97 kd phosphoprotein contained 39% phosphoserine (p-ser) and 61% phosphothreonine (p-thr); and the 55 kd phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr). The kinases acting on all four phosphoproteins were independent of Ca 2+ , cAMP, cGMP, or phorbol 12-myristate 13-acetate. The observed differences in phosphorylation events between sparse and confluent membranes occurred in membranes from two EC lines - pig aortic and bovine aortic - but were not apparent in membranes prepared from human foreskin fibroblasts or 3T3 cells. Sparse endothelial cells made quiescent by serum deprivation were found to resemble confluent cells in the kinase activity; therefore, the enhanced kinase activity in sparse membranes may be growth dependent

Magnetizable stent-grafts enable endothelial cell capture

Science.gov (United States)

Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

2017-04-01

Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

Directory of Open Access Journals (Sweden)

Kyle J Hewitt

2011-02-01

Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

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Eva C. Thoma

2014-10-01

Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.

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Pincha, Neha; Hajam, Edries Yousaf; Badarinath, Krithika; Batta, Surya Prakash Rao; Masudi, Tafheem; Dey, Rakesh; Andreasen, Peter; Kawakami, Toshiaki; Samuel, Rekha; George, Renu; Danda, Debashish; Jacob, Paul Mazhuvanchary; Jamora, Colin

2018-05-01

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

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Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

2017-11-01

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

African Journals Online (AJOL)

induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

Effects of endothelial cells on human mesenchymal stem cell activity in a three-dimensional in vitro model

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A Saleh

2011-10-01

Full Text Available An increasing body of data suggest that mesenchymal stem cells (MSCs reside in a perivascular niche. To more closely mimic this in vivo microenvironment and for better understanding of its complexity, and the factors that regulate the MSC activity, human umbilical vein endothelial cells (HUVECs were co-cultured with human bone marrow MSCs – using a novel three-dimensional (3D spheroid co-culture system. Using confocal microscopy of fluorescently labelled cells, we observed HUVECs and MSCs to self-assemble and form organised structures with segregated cell-type partitioning. Under osteogenic conditions, the rate and extent of differentiation in MSC/HUVEC spheroids was significantly elevated compared to 3D co-cultures of MSCs and human dermal fibroblast controls as shown by alkaline phosphatase staining. Conversely, HUVECs inhibited adipogenic differentiation and the proliferation of MSCs in 3D co-cultures indicating that HUVECs suppressed MSC cycling and selectively promoted osteogenic differentiation in 3D. We have also shown that HUVECs enhanced activation of endogenous Wnt signalling and bone morphogenetic protein (BMP signalling as shown by increased levels of active nuclear β-catenin and pSmad 1/5/8 immunopositivity respectively. These data suggest strongly that endothelial cells regulate the MSC activity in simulated in vivo conditions, by maintaining quiescence and facilitating niche exit via osteogenic differentiation following appropriate cues. Our findings also underline the importance of 3D heterotypic cell-cell interactions in the regulation of MSC behaviour, suggesting that multicellular cocktails and/or 3D-based delivery strategies may be beneficial for bone repair.

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

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Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

2017-04-06

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

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Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-11-01

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and

Infection of endothelial cells by common human viruses.

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Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

[Expression profiles and bioinformatic analysis of miRNA in human dental pulp cells during endothelial differentiation].

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Gong, Qimei; Jiang, Hongwei; Wang, Jinming; Ling, Junqi

2014-05-01

To investigate the differential expression profile and bioinformatic analysis of microRNA (miRNA) in human dental pulp cells (DPC) during endothelial differentiation. DPC were cultured in endothelial induction medium (50 µg/L vascular endothelial growth factor, 10 µg/L basic fibroblast growth factor and 2% fetal calf serum) for 7 days. Meanwhile non-induced DPC were used as control.Quantitative real-time PCR (qRT-PCR) was applied to detect vascular endothelial marker genes [CD31, von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin)] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells. And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR. Furthermore, bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction. The relative mRNA level of CD31, vWF and VE-cadherin in the control group were (3.48 ± 0.22) ×10(-4), (3.13 ± 0.31) ×10(-4) and (39.60 ± 2.36) ×10(-4), and (19.57 ± 2.20) ×10(-4), (48.13 ± 0.54) ×10(-4) and (228.00 ± 8.89) ×10(-4) in the induced group. The expressions of CD31, vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group (P functions, such as the regulation of transcription, cell motion, blood vessel morphogenesis, angiogenesis and cytoskeletal protein, and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway. The differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation, thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells.

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Fletcher, Anne L; Malhotra, Deepali; Acton, Sophie E; Lukacs-Kornek, Veronika; Bellemare-Pelletier, Angelique; Curry, Mark; Armant, Myriam; Turley, Shannon J

2011-01-01

Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs) between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

The effects of acoustic vibration on fibroblast cell migration.

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Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

2016-12-01

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

Signaling hierarchy regulating human endothelial cell development

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Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

International Nuclear Information System (INIS)

Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

2007-01-01

Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain

Animal study on transplantation of human umbilical vein endothelial cells for corneal endothelial decompensation

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Li Cui

2014-06-01

Full Text Available AIM: To explore the feasibility of culturing human umbilical vein endothelial cells(HUVECon acellular corneal stroma and performing the posterior lamellar endothelial keratoplasty(PLEKtreating corneal endothelial decompensation.METHODS: Thirty New-Zealand rabbits were divided into three groups randomly, 10 rabbits for experimental group, 10 for stroma group and 10 for control group. Corneal endothelial cells were removed to establish animal model of corneal endothelial failure. PLEK was performed on the rabbits of experimental group and stroma group, and nothing was transplantated onto the rabbits of control group with the deep layer excised only. Postoperative observation was taken for 3mo. The degree of corneal edema and central corneal thickness were recorded for statistical analysis.RESULTS: Corneas in experimental group were relieved in edema obviously compared with that in stroma group and the control group, and showed increased transparency 7d after the operation. The average density of endothelial cells was 2 026.4±129.3cells/mm2, and average central corneal thickness was 505.2±25.4μm in experimental group, while 1 535.6±114.5μm in stroma group and 1 493.5±70.2μm in control group 3mo after operation.CONCLUSION:We achieved preliminary success in our study that culturing HUVEC on acellular corneal stroma and performing PLEK for corneal endothelial decompensation. HUVEC transplanted could survive in vivo, and have normal biological function of keeping cornea transparent. This study provides a new idea and a new way clinically for the treatment of corneal endothelial diseases.

Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells

International Nuclear Information System (INIS)

Matayoshi, Takaaki; Goto, Tetsuya; Fukuhara, Eiji; Takano, Hiroshi; Kobayashi, Shigeru; Takahashi, Tetsu

2005-01-01

The present study was designed to evaluate the effects of neuropeptide substance P (Sp) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the Sp receptor, neurokinin-1 receptor (NK 1 -R). The addition of Sp stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by Sp or NK 1 -R antagonists. Increased expression of the receptor activator of nuclear factor κB ligand (Rankle) in synovial fibroblastic cells after the addition of Sp was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium

Cytotoxicity of four denture adhesives on human gingival fibroblast cells.

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Lee, Yoon; Ahn, Jin-Soo; Yi, Young-Ah; Chung, Shin-Hye; Yoo, Yeon-Jee; Ju, Sung-Won; Hwang, Ji-Yun; Seo, Deog-Gyu

2015-02-01

The purpose of this study was to compare the cytotoxicity of four denture adhesives on human gingival fibroblast cells. Immortalized human gingival fibroblasts were cultured with one of four different denture adhesives, Polident, Protefix, Staydent or Denfix-A, which was placed in insert dishes (10% w/v concentration) for 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometric apoptosis assay were used to evaluate cell viability and apoptosis rates. The fibroblasts were also examined under a scanning electron microscope. The MTT assay showed that all denture adhesives resulted in a significantly lower cell viability compared to the control cells propagated in normal culture medium (p 0.05). Staydent showed the highest apoptosis rate. Scanning electron microscopy showed that the cells of the Staydent group underwent cytoplasmic membrane shrinkage, with cell free areas containing residual fragments of the membrane of dead cells. The four denture adhesives evaluated in this study imparted cytotoxic effects on human gingival fibroblast cells. Staydent showed the highest toxicity.

Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts.

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Liu, Wei; Krump, Nathan A; MacDonald, Margo; You, Jianxin

2018-02-15

Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), rat ( Rattus norvegicus ), chimpanzee ( Pan troglodytes ), rhesus macaque ( Macaca mulatta ), patas monkey ( Erythrocebus patas ), common woolly monkey ( Lagothrix lagotricha ), red-chested mustached tamarin ( Saguinus labiatus ), and tree shrew ( Tupaia belangeri ). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression. IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel

Transport of lipoprotein lipase across endothelial cells

International Nuclear Information System (INIS)

Saxena, U.; Klein, M.G.; Goldberg, I.J.

1991-01-01

Lipoprotein lipase (LPL), synthesized in muscle and fat, hydrolyzes plasma triglycerides primarily while bound to luminal endothelial cell surfaces. To obtain information about the movement of LPL from the basal to the luminal endothelial cell surface, the authors studied the transport of purified bovine milk LPL across bovine aortic endothelial cell monolayers. 125 I-labeled LPL ( 125 I-LPL) added to the basal surface of the monolayers was detected on the apical side of the cells in two compartments: (1) in the medium of the upper chamber, and (2) bound to the apical cell surface. The amount of 125 I-LPL on the cell surface, but not in the medium, reached saturation with time and LPL dose. Catalytically active LPL was transported to the apical surface but very little LPL activity appeared in the medium. Heparinase treatment of the basal cell surface and addition of dextran sulfate to the lower chamber decreased the amount of 125 I-LPL appearing on the apical surface. Similarly, the presence of increasing molar ratios of oleic acid/bovine serum albumin at the basal surface decreased the transport of active LPL across the monolayer. Thus, a saturable transport system, which requires haparan sulfate proteoglycans and is inhibited by high concentrations of free fatty acids on the basal side of the cells, appears to exist for passage of enzymatically active LPL across endothelial cells. They postulate that regulation of LPL transport to the endothelial luminal surface modulates the physiologically active pool of LPL in vivo

Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.

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Langan, Stacey A; Navarro-Núñez, Leyre; Watson, Steve P; Nash, Gerard B

2017-07-20

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

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Magnusson Magnus K

2010-07-01

Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

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Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Identification and functional analysis of endothelial tip cell-enriched genes.

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del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef; Eichmann, Anne

2010-11-11

Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

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Sakai, Norihiko; Tager, Andrew M.

2013-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

DEFF Research Database (Denmark)

Wang, X; Häring, M-F; Rathjen, Thomas

2017-01-01

in vascular endothelial cells. Strikingly, these mice had 42% more intestinal tumours than controls, no change in tumour angiogenesis, but increased expression of vascular cell adhesion molecule-1 (VCAM-1) in primary culture of tumour endothelial cells. Insulin decreased VCAM-1 expression and leukocyte...... adhesion in quiescent tumour endothelial cells with intact insulin receptors and partly prevented increases in VCAM-1 and leukocyte adhesion after treatment with tumour necrosis factor-α. Knockout of insulin receptors in endothelial cells also increased leukocyte adhesion in mesenteric venules...

Modeling triple-negative breast cancer heterogeneity: effects of stromal macrophages, fibroblasts and tumor vasculature.

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Norton, Kerri-Ann; Jin, Kideok; Popel, Aleksander S

2018-05-08

A hallmark of breast tumors is its spatial heterogeneity that includes its distribution of cancer stem cells and progenitor cells, but also heterogeneity in the tumor microenvironment. In this study we focus on the contributions of stromal cells, specifically macrophages, fibroblasts, and endothelial cells on tumor progression. We develop a computational model of triple-negative breast cancer based on our previous work and expand it to include macrophage infiltration, fibroblasts, and angiogenesis. In vitro studies have shown that the secretomes of tumor-educated macrophages and fibroblasts increase both the migration and proliferation rates of triple-negative breast cancer cells. In vivo studies also demonstrated that blocking signaling of selected secreted factors inhibits tumor growth and metastasis in mouse xenograft models. We investigate the influences of increased migration and proliferation rates on tumor growth, the effect of the presence on fibroblasts or macrophages on growth and morphology, and the contributions of macrophage infiltration on tumor growth. We find that while the presence of macrophages increases overall tumor growth, the increase in macrophage infiltration does not substantially increase tumor growth and can even stifle tumor growth at excessive rates. Copyright © 2018. Published by Elsevier Ltd.

Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

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Der-I Kao

2015-02-01

Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

Extraembryonic origin of circulating endothelial cells.

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Luc Pardanaud

Full Text Available Circulating endothelial cells (CEC are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

Extraembryonic origin of circulating endothelial cells.

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Pardanaud, Luc; Eichmann, Anne

2011-01-01

Circulating endothelial cells (CEC) are contained in the bone marrow and peripheral blood of adult humans and participate to the revascularization of ischemic tissues. These cells represent attractive targets for cell or gene therapy aimed at improving ischemic revascularization or inhibition of tumor angiogenesis. The embryonic origin of CEC has not been addressed previously. Here we use quail-chick chimeras to study CEC origin and participation to the developing vasculature. CEC are traced with different markers, in particular the QH1 antibody recognizing only quail endothelial cells. Using yolk-sac chimeras, where quail embryos are grafted onto chick yolk sacs and vice-versa, we show that CEC are generated in the yolk sac. These cells are mobilized during wound healing, demonstrating their participation to angiogenic repair processes. Furthermore, we found that the allantois is also able to give rise to CEC in situ. In contrast to the yolk sac and allantois, the embryo proper does not produce CEC. Our results show that CEC exclusively originate from extra-embryonic territories made with splanchnopleural mesoderm and endoderm, while definitive hematopoietic stem cells and endothelial cells are of intra-embryonic origin.

Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

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S Montagnani

2009-12-01

Full Text Available Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

Reproducible isolation of lymph node stromal cells reveals site-dependent differences in fibroblastic reticular cells

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Anne L Fletcher

2011-09-01

Full Text Available Within lymph nodes, non-hematopoietic stromal cells organize and interact with leukocytes in an immunologically important manner. In addition to organizing T and B cell segregation and expressing lymphocyte survival factors, several recent studies have shown that lymph node stromal cells shape the naïve T cell repertoire, expressing self-antigens which delete self-reactive T cells in a unique and non-redundant fashion. A fundamental role in peripheral tolerance, in addition to an otherwise extensive functional portfolio, necessitates closer study of lymph node stromal cell subsets using modern immunological techniques; however this has not routinely been possible in the field, due to difficulties reproducibly isolating these rare subsets. Techniques were therefore developed for successful ex vivo and in vitro manipulation and characterization of lymph node stroma. Here we discuss and validate these techniques in mice and humans, and apply them to address several unanswered questions regarding lymph node composition. We explored the steady-state stromal composition of lymph nodes isolated from mice and humans, and found that marginal reticular cells and lymphatic endothelial cells required lymphocytes for their normal maturation in mice. We also report alterations in the proportion and number of fibroblastic reticular cells (FRCs between skin-draining and mesenteric lymph nodes. Similarly, transcriptional profiling of FRCs revealed changes in cytokine production from these sites. Together, these methods permit highly reproducible stromal cell isolation, sorting, and culture.

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

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Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

2013-01-01

The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Functional and gene expression analysis of hTERT overexpressed endothelial cells

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Haruna Takano

2008-09-01

Full Text Available Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

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Yulia Shamis

Full Text Available Human embryonic and induced pluripotent stem cells (hESC/hiPSC are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.

5-Hydroxytryptamine 4 Receptor in the Endothelial Cells

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Profirovic, Jasmina; Vardya, Irina; Voyno-Yasenetskaya, Tatyana

2006-01-01

39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events in the ce......39 5-HYDROXYTRYPTAMINE 4 RECEPTOR IN THE ENDOTHELIAL CELLS. J. Profirovic, I. Vardya, T. Voyno-Yasenetskaya, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL. Serotonin (5-hydroxytryptamine [5-HT]) is an important neurotransmitter that regulates multiple events...... gap formation in HUVECs. We are currently investigating the mechanism underlying 5-HT4 receptor-induced actin cytoskeleton changes in the endothelial cells. These data suggest that by activating 5-HT4 receptor, serotonin could be involved in regulation of actin cytoskeleton dynamics in the endothelial...

Vascular endothelial growth factor modified macrophages transdifferentiate into endothelial-like cells and decrease foam cell formation.

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Yan, Dan; He, Yujuan; Dai, Jun; Yang, Lili; Wang, Xiaoyan; Ruan, Qiurong

2017-06-30

Macrophages are largely involved in the whole process of atherosclerosis from an initiation lesion to an advanced lesion. Endothelial disruption is the initial step and macrophage-derived foam cells are the hallmark of atherosclerosis. Promotion of vascular integrity and inhibition of foam cell formation are two important strategies for preventing atherosclerosis. How can we inhibit even the reverse negative role of macrophages in atherosclerosis? The present study was performed to investigate if overexpressing endogenous human vascular endothelial growth factor (VEGF) could facilitate transdifferentiation of macrophages into endothelial-like cells (ELCs) and inhibit foam cell formation. We demonstrated that VEGF-modified macrophages which stably overexpressed human VEGF (hVEGF 165 ) displayed a high capability to alter their phenotype and function into ELCs in vitro Exogenous VEGF could not replace endogenous VEGF to induce the transdifferentiation of macrophages into ELCs in vitro We further showed that VEGF-modified macrophages significantly decreased cytoplasmic lipid accumulation after treatment with oxidized LDL (ox-LDL). Moreover, down-regulation of CD36 expression in these cells was probably one of the mechanisms of reduction in foam cell formation. Our results provided the in vitro proof of VEGF-modified macrophages as atheroprotective therapeutic cells by both promotion of vascular repair and inhibition of foam cell formation. © 2017 The Author(s).

PECAM-1 polymorphism affects monocyte adhesion to endothelial cells.

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Goodman, Reyna S; Kirton, Christopher M; Oostingh, Gertie J; Schön, Michael P; Clark, Michael R; Bradley, J Andrew; Taylor, Craig J

2008-02-15

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) plays an important role in leukocyte-endothelial cell adhesion and transmigration. Single nucleotide polymorphisms of PECAM-1 encoding amino acid substitutions at positions 98 leucine/valine (L/V), 536 serine/asparagine (S/N), and 643 arginine/glycine (R/G) occur in strong genetic linkage resulting in two common haplotypes (LSR and VNG). These PECAM-1 polymorphisms are associated with graft-versus-host disease after hematopoietic stem cell transplantation and with cardiovascular disease, but whether they influence PECAM-1 function is unknown. We examined the effect of homozygous and heterozygous expression of the PECAM-1 LSR and VNG genotypes on the adhesive interactions of peripheral blood monocytes and activated endothelial cell monolayers under shear stress in a flow-based cell adhesion assay. There was no difference in monocyte adhesion between the two homozygous genotypes of PECAM-1 but when monocytes expressed both alleles in heterozygous form, firm adhesion of monocytes to endothelial cells was markedly increased. PECAM-1 polymorphism expressed in homozygous or heterozygous form by endothelial cells did not influence monocyte adhesion. This is, to our knowledge, the first demonstration that PECAM-1 genotype can alter the level of monocyte binding to endothelial cells and a demonstration that heterozygous expression of a polymorphic protein may lead to altered function.

Adipose stem cells for bone tissue repair

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Ciuffi, Simone; Zonefrati, Roberto; Brandi, Maria Luisa

2017-01-01

Adipose-derived stem/stromal cells (ASCs), together with adipocytes, vascular endothelial cells, and vascular smooth muscle cells, are contained in fat tissue. ASCs, like the human bone marrow stromal/stem cells (BMSCs), can differentiate into several lineages (adipose cells, fibroblast, chondrocytes, osteoblasts, neuronal cells, endothelial cells, myocytes, and cardiomyocytes). They have also been shown to be immunoprivileged, and genetically stable in long-term cultures. Nevertheless, unlik...

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects.

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Bhadra, S; Arshad, M A; Rymaszewski, Z; Norman, E; Wherley, R; Subbiah, M T

1991-04-15

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.

Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

International Nuclear Information System (INIS)

Borek, C.

1982-01-01

In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

Resveratrol induces mitochondrial biogenesis in endothelial cells.

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Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

2009-07-01

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

Ionizing radiation activates vascular endothelial growth factor-A transcription in human umbilical vein endothelial cells

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Lee, Hyounji; Kim, Kwang Seok; Jeong, Jae Hoon; Lim, Young Bin [Radiation Cancer Biology Team, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2016-12-15

Vascular endothelial growth factor (VEGF) is an essential paracrine factor for developmental and pathological angiogenesis. VEGF also exerts its effects in an autocrine manner in VEGF-producing cells. For instance, autocrine VEGF signaling occurs in tumor cells and contributes to key aspects of tumorigenesis, such as in the function of cancer stem cells and tumor initiation, which are independent of angiogenesis. In addition to tumors cells, non-transformed cells also express VEGF. For example, a VEGF dependent intracellular autocrine mechanism is crucial for the survival of hematopoietic stem cells and hematopoiesis. Stereotactic body radiation therapy (SBRT) is a novel treatment modality for early primary cancer and oligometastatic disease. SBRT delivers high-dose hypofractionated radiation, such as 20-60 Gy, to tumors in a single fraction or 2-5 fractions. As VEGF is a critical regulator of functional integrity and viability of vascular endothelial cells, we examined whether high-dose irradiation alters VEGF signaling by measuring the expression levels of VEGFA transcript. It is generally believed that endothelial cells do not produce VEGF in response to radiation. In present study, however, we provide the first demonstration of transcriptional regulation of VEGFA in human vascular endothelial cells by IR treatment. Irradiation with doses higher than 10 Gy in a single exposure triggers up-regulation of VEGFA transcription within 2 hours in HUVECs, whereas irradiation with 10 Gy does not alter VEGFA levels. Our data have shown that high-dose irradiation triggers immediate transactivation of VEGFA in human vascular endothelial cells.

Orf virus interleukin-10 and vascular endothelial growth factor-E modulate gene expression in cultured equine dermal fibroblasts.

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Wise, Lyn M; Bodaan, Christa J; Mercer, Andrew A; Riley, Christopher B; Theoret, Christine L

2016-10-01

Wounds in horses often exhibit sustained inflammation and inefficient vascularization, leading to excessive fibrosis and clinical complications such as "proud flesh". Orf virus-derived proteins, vascular endothelial growth factor (VEGF)-E and interleukin (ovIL)-10, enhance angiogenesis and control inflammation and fibrosis in skin wounds of laboratory animals. The study aimed to determine if equine dermal cells respond to VEGF-E and ovIL-10. Equine dermal cells are expected to express VEGF and IL-10 receptors, so viral protein treatment is likely to alter cellular gene expression and behaviour in a manner conducive to healing. Skin samples were harvested from the lateral thoracic wall of two healthy thoroughbred horses. Equine dermal cells were isolated using a skin explant method and their phenotype assessed by immunofluorescence. Cells were treated with recombinant proteins, with or without inflammatory stimuli. Gene expression was examined using standard and quantitative reverse transcriptase PCR. Cell behaviour was evaluated in a scratch assay. Cultured cells were half vimentin(+ve) fibroblasts and half alpha smooth muscle actin(+ve) and vimentin(+ve) myofibroblasts. VEGF-E increased basal expression of IL-10 mRNA, whereas VEGF-A and collagenase-1 mRNA expression was increased by ovIL-10. In cells exposed to inflammatory stimulus, both treatments dampened tumour necrosis factor mRNA expression, and ovIL-10 exacerbated expression of monocyte chemoattractant protein. Neither viral protein influenced cell migration greatly. This study shows that VEGF-E and ovIL-10 are active on equine dermal cells and exert anti-inflammatory and anti-fibrotic effects that may enhance skin wound healing in horses. © 2016 ESVD and ACVD.

Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

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Kathryn L McCabe

Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

Lipoprotein receptors in cultured bovine endothelial cells

International Nuclear Information System (INIS)

Struempfer, A.E.M.

1983-07-01

In this study, receptors that may be involved in the uptake of low density lipoproteins (LDL) and low density lipoproteins which have been modified by acetylation (AcLDL), were characterized. Aortic epithelial cells were used and a cell culture system which closely resembled the in vivo monolayer was established. Endothelial cell and lipoprotein interactions were examined by incubating the cells with 125 l-labelled lipoproteins under various conditions. The receptor affinity of bovine aortic endothelial cells was higher for AcLDL than that for LDL. Competition studies demonstrated that there were two distinct receptors for LDL and AcLDL on the endothelial cells. AcLDL did not compete with LDL for the LDL receptor, and conversely LDL did not compete with AcLDL for the AcLDL receptor. The receptor activities for LDL and AcLDL were examined as a function of culture age. Whereas the LDL receptor could be regulated, the AcLDL receptor was not as susceptible to regulation. Upon exposing endothelial cells for 72 h to either LDL or AcLDL, it was found that the total amount of cellular cholesterol increased by about 50%. However, the increase of total cholesterol was largely in the form of free cholesterol. This is in contrast to macrophages, where the increase in total cholesterol upon exposure to AcLDL is largely in the form cholesteryl esters

The Expression Profiles of Lysophospholipid Receptors (LPLRs in Different Endothelial Cells

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Yu-Wei Lee

2006-03-01

Full Text Available Sphingosine-1-phosphate (S1P and lysophosphatidic acid (LPA are two bioactive lysophospholipids (LPLs, stored primarily in platelets and released during platelet activation. Both LPLs are capable of regulating endothelial cell functions. The physiological functions of S1P and LPA are mediated by interacting with eight different G-protein coupled receptors: S1P1 through 5 and LPA1 through 3, which activate three different heterotrimeric GTP proteins－including Gi、Gq and G(12/13. The expression of LPL receptors in endothelial cells would affect the responses of S1P and LPA to these cells. There is no previous report discussing the expression profiles of LPL receptors in different endothelial cells from various species. In this study, we aim to investigate the expression profiles of S1P and LPA receptors in different endothelial cells isolated from human, rat, mouse and bovine origin. We used RT-PCR to determine LPLs receptors expression profiles in different endothelial cells. Our results indicated that endothelial cells from various species express different LPL receptors. Endothelial cells isolated from the same source of different species also had different LPLs receptors expression profiles. Therefore, different endothelial cells should respond to LPLs in different manners.

Culture and Characterization of Circulating Endothelial Progenitor Cells in Patients with Renal Cell Carcinoma.

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Gu, Wenyu; Sun, Wei; Guo, Changcheng; Yan, Yang; Liu, Min; Yao, Xudong; Yang, Bin; Zheng, Junhua

2015-07-01

Although emerging evidence demonstrates increased circulating endothelial progenitor cells in patients with solid tumors, to our knowledge it is still unknown whether such cells can be cultured from patients with highly angiogenic renal cell carcinoma. We cultured and characterized circulating endothelial progenitor cells from patients with renal cell carcinoma. The circulating endothelial progenitor cell level (percent of CD45(-)CD34(+) VEGF-R2(+) cells in total peripheral blood mononuclear cells) was quantified in 47 patients with renal cell carcinoma and 40 healthy controls. Peripheral blood mononuclear cells were then isolated from 33 patients with renal cell carcinoma and 30 healthy controls to culture and characterize circulating endothelial progenitor cells. The circulating endothelial progenitor cell level was significantly higher in patients with renal cell carcinoma than in healthy controls (0.276% vs 0.086%, p cells first emerged significantly earlier in patient than in control preparations (6.72 vs 14.67 days, p culture success rate (87.8% vs 40.0% of participants) and the number of colonies (10.06 vs 1.83) were significantly greater for patients than for controls (each p cell level correlated positively with the number of patient colonies (r = 0.762, p Cells cultured from patients and controls showed a similar growth pattern, immunophenotype, ability to uptake Ac-LDL and bind lectin, and form capillary tubes in vitro. However, significantly more VEGF-R2(+) circulating endothelial progenitor cells were found in preparations from patients with renal cell carcinoma than from healthy controls (21.1% vs 13.4%, p cell colonies, a higher cell culture success rate and more colonies were found for patients with renal cell carcinoma than for healthy controls. Results indicate the important significance of VEGF-R2(+) circulating endothelial progenitors in patients with renal cell carcinoma. Copyright © 2015 American Urological Association Education and Research

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

Science.gov (United States)

Jansen, Felix; Yang, Xiaoyan; Hoelscher, Marion; Cattelan, Arianna; Schmitz, Theresa; Proebsting, Sebastian; Wenzel, Daniela; Vosen, Sarah; Franklin, Bernardo S; Fleischmann, Bernd K; Nickenig, Georg; Werner, Nikos

2013-10-29

Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Endothelial Induced EMT in Breast Epithelial Cells with Stem Cell Properties

OpenAIRE

Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla; Fridriksdottir, Agla J. R.; Ringnér, Markus; Villadsen, Rene; Borg, Ake; Agnarsson, Bjarni A.; Petersen, Ole William; Magnusson, Magnus K.; Gudjonsson, Thorarinn

2011-01-01

Epithelial to mesenchymal transition (EMT) is a critical event in cancer progression and is closely linked to the breast epithelial cancer stem cell phenotype. Given the close interaction between the vascular endothelium and cancer cells, especially at the invasive front, we asked whether endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell proper...

Tumor-derived circulating endothelial cell clusters in colorectal cancer.

KAUST Repository

Cima, Igor; Kong, Say Li; Sengupta, Debarka; Tan, Iain B; Phyo, Wai Min; Lee, Daniel; Hu, Min; Iliescu, Ciprian; Alexander, Irina; Goh, Wei Lin; Rahmani, Mehran; Suhaimi, Nur-Afidah Mohamed; Vo, Jess H; Tai, Joyce A; Tan, Joanna H; Chua, Clarinda; Ten, Rachel; Lim, Wan Jun; Chew, Min Hoe; Hauser, Charlotte; van Dam, Rob M; Lim, Wei-Yen; Prabhakar, Shyam; Lim, Bing; Koh, Poh Koon; Robson, Paul; Ying, Jackie Y; Hillmer, Axel M; Tan, Min-Han

2016-01-01

Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Tumor-derived circulating endothelial cell clusters in colorectal cancer.

KAUST Repository

Cima, Igor

2016-06-29

Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Viability test of fish scale collagen (Oshpronemus gouramy on baby hamster kidney fibroblasts-21 fibroblast cell culture

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Chiquita Prahasanti

2018-04-01

Full Text Available Aim: This study aims to examine the toxicity of collagen extracted from gouramy fish scales (Oshpronemus gouramy by evaluating its viability against baby hamster kidney fibroblasts-21. Materials and Methods: Collagen was extracted from gouramy fish scales (O. gouramy with 6% acetic acid. Its results were analyzed using Fourier-transform infrared spectroscopy and freeze-dried technique. Its morphology then was analyzed with scanning electron microscope. Afterward, 3-(4.5-dimethylthiazole-2-yl2.5-diphenyl tetrazolium bromide assay was conducted to compare cells with and without fish scale collagen treatment. Results: Collagen extracted from gouramy fish scales had no influence statistically on cultured fibroblast cells with a statistical significance (2-tailed value of 0.754 (p>00025. Conclusion: Collagen extracted from gouramy fish scales has high viability against BHK21 fibroblast cells.

circHECTD1 promotes the silica-induced pulmonary endothelial-mesenchymal transition via HECTD1.

Science.gov (United States)

Fang, Shencun; Guo, Huifang; Cheng, Yusi; Zhou, Zewei; Zhang, Wei; Han, Bing; Luo, Wei; Wang, Jing; Xie, Weiping; Chao, Jie

2018-03-14

Excessive proliferation and migration of fibroblasts contribute to pulmonary fibrosis in silicosis, and both epithelial cells and endothelial cells participate in the accumulation of fibroblasts via the epithelial-mesenchymal transition (EMT) and the endothelial-mesenchymal transition (EndMT), respectively. A mouse endothelial cell line (MML1) was exposed to silicon dioxide (SiO 2 , 50 μg/cm 2 ), and immunofluorescence and western blot analyses were performed to evaluate levels of specific endothelial and mesenchymal markers and to elucidate the mechanisms by which SiO 2 induces the EndMT. Functional changes were evaluated by analyzing cell migration and proliferation. The mRNA and circular RNA (circRNA) levels were measured using qPCR and fluorescent in situ hybridization (FISH). Lung tissue samples from both Tie2-GFP mice exposed to SiO 2 and silicosis patients were applied to confirm the observations from in vitro experiments. Based on the results from the current study, SiO 2 increased the expression of mesenchymal markers (type I collagen (COL1A1), type III collagen (COL3A1) and alpha smooth muscle actin (α-SMA/Acta2)) and decreased the expression of endothelial markers (vascular endothelial cadherin (VE-Cad/Cdh 5) and platelet endothelial cell adhesion molecule-1 (PECAM1)), indicating the occurrence of the EndMT in response to SiO 2 exposure both in vivo and in vitro. SiO 2 concomitantly increased circHECTD1 expression, which, in turn, inhibited HECTD1 protein expression. SiO 2 -induced increases in cell proliferation, migration, and changes in marker levels were restored by either a small interfering RNA (siRNA) targeting circHECTD1 or overexpression of HECTD1 via the CRISPR/Cas9 system, confirming the involvement of the circHECTD1/HECTD1 pathway in the EndMT. Moreover, tissue samples from SiO 2 -exposed mice and silicosis patients confirmed the EndMT and change in HECTD1 expression. Our findings reveal a potentially new function for the circHECTD1/HECTD

Effect of bFGF on radiation-induced apoptosis of vascular endothelial cells

International Nuclear Information System (INIS)

Gu Qingyang; Wang Dewen; Li Yuejuan; Peng Ruiyun; Dong Bo; Wang Zhaohai; Liu Jie; Deng Hua; Jiang Tao

2003-01-01

Objective: To study the effect of bFGF on radiation-induced apoptosis vascular endothelial cells. Methods: A cell line PAE (porcine aortic endothelial cells) and primary cultured HUVEC (human umbilical vein endothelial cells) were irradiated with 60 Co γ-rays to establish cell apoptosis models. Flow cytometry with annexin-V-FITC + PI labeling was used to evaluate cell apoptosis. Different amounts of bFGF were used to study their effects on radiation-induced endothelial cell apoptosis. Results and Conclusions: It is found that bFGF could inhibit radiation-induced endothelial cell apoptosis in a considerable degree

Irradiated murine fibroblasts as feeder layer used in human cell culture

International Nuclear Information System (INIS)

Almeida, Tiago L.; Klingbeil, Fatima G.; Yoshito, Daniele; Caproni, Priscila; Mathor, Monica B.; Herson, Marisa R.

2007-01-01

In 1975, Rheinwald and Green published an in vitro model for keratinocyte cell cultures in which the use of murine fibroblasts, as a feeder layer was introduced. These cells are modified fibroblasts, which presence render keratinocyte cells to remain proliferative for longer periods of time. This optimization of culture outputs has allowed for several clinical applications of confluent keratinocyte cultures as skin substitutes or wound dressings in situations such as post burn extensive skin loss, loss of oral mucosa, and other skin disorders. Nevertheless, proliferation of fibroblast in co-culture with keratinocytes must be controlled by anti-proliferative measures such as irradiation; at the same time, keratinocytes require specific nutrients in the culture medium, which may interfere with the fibroblast feeder layer viability. Therefore, the thorough understanding of the impact of different issues such as culture media composition, irradiation dose and pre-plating storage conditions of irradiated fibroblast to be used as feeder layer in these co-culture systems is important. In this work, changes as far as viability and proliferative rates of irradiated fibroblasts in culture were evaluated in relation to the type of culture medium used, dose of gamma radiation exposure, storage and timing of cell plating post irradiation. Results indicate that the type of culture medium used and time-lag between irradiation, refrigeration and plating of irradiated cells do not have significant impact in culture outcomes. However, the dose of gamma radiation administered to the cells may influence the final quality of these cells if to be used as a feeder layer. (author)

The influence of biomaterials on endothelial cell thrombogenicity

Science.gov (United States)

McGuigan, Alison P.; Sefton, Michael V.

2007-01-01

Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their non-thrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thromobgenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions. PMID:17316788

Endothelial cell proliferation in swine experimental aneurysm after coil embolization.

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Yumiko Mitome-Mishima

Full Text Available After coil embolization, recanalization in cerebral aneurysms adversely influences long-term prognosis. Proliferation of endothelial cells on the coil surface may reduce the incidence of recanalization and further improve outcomes after coil embolization. We aimed to map the expression of proliferating tissue over the aneurysmal orifice and define the temporal profile of tissue growth in a swine experimental aneurysm model. We compared the outcomes after spontaneous thrombosis with those of coil embolization using histological and morphological techniques. In aneurysms that we not coiled, spontaneous thrombosis was observed, and weak, easily detachable proliferating tissue was evident in the aneurysmal neck. In contrast, in the coil embolization group, histological analysis showed endothelial-like cells lining the aneurysmal opening. Moreover, immunohistochemical and morphological analysis suggested that these cells were immature endothelial cells. Our results indicated the existence of endothelial cell proliferation 1 week after coil embolization and showed immature endothelial cells in septal tissue between the systemic circulation and the aneurysm. These findings suggest that endothelial cells are lead to and proliferate in the former aneurysmal orifice. This is the first examination to evaluate the temporal change of proliferating tissue in a swine experimental aneurysm model.

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

Energy Technology Data Exchange (ETDEWEB)

Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

2011-10-07

Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

International Nuclear Information System (INIS)

Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

2011-01-01

Highlights: → Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. → Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. → We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. → Collagen type I and collagen type III mRNA level was higher in differentiated cells. → UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.

CORNEAL ENDOTHELIAL CELL DENSITY IN ACUTE ANGLE CLOSURE GLAUCOMA

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Nishat Sultana K

2016-09-01

Full Text Available BACKGROUND Angle closure is characterised by apposition of the peripheral iris against the trabecular meshwork resulting in obstruction of aqueous outflow. Acute angle-closure glaucoma is characterised by pain, redness and blurred vision. The pain is typically a severe deep ache that follows the trigeminal distribution and maybe associated with nausea, vomiting, bradycardia and profuse sweating. The blurred vision, which is typically marked maybe caused by stretching of the corneal lamellae initially and later oedema of the cornea as well as a direct effect of the IOP on the optic nerve head. The modifications in corneal endothelial cell density after a crisis of angle-closure glaucoma is being evaluated. AIMS AND OBJECTIVES The objective of the study is to assess the corneal endothelial cell count (density by specular microscopy in patients presenting with acute angle-closure glaucoma. METHODS Corneal endothelial cell counts of 20 eyes of patients with PACG with an earlier documented symptomatic acute attack unilaterally were compared with 20 fellow eyes. Evaluation of patient included visual acuity, intraocular pressure, gonioscopy, disc findings and specular microscopy. RESULTS The mean endothelial cell density was 2104 cells/mm2 in the eye with acute attack and 2615 cells/mm2 in the fellow eye. The average endothelial cell count when the duration of attack lasted more than 72 hours was 1861 cells/mm2 . CONCLUSION Corneal endothelial cell density was found to be significantly reduced in eyes following an acute attack of primary angle closure glaucoma.

A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

DEFF Research Database (Denmark)

Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

1992-01-01

major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

Blood on the tracks: hematopoietic stem cell-endothelial cell interactions in homing and engraftment.

Science.gov (United States)

Perlin, Julie R; Sporrij, Audrey; Zon, Leonard I

2017-08-01

Cells of the hematopoietic system undergo rapid turnover. Each day, humans require the production of about one hundred billion new blood cells for proper function. Hematopoietic stem cells (HSCs) are rare cells that reside in specialized niches and are required throughout life to produce specific progenitor cells that will replenish all blood lineages. There is, however, an incomplete understanding of the molecular and physical properties that regulate HSC migration, homing, engraftment, and maintenance in the niche. Endothelial cells (ECs) are intimately associated with HSCs throughout the life of the stem cell, from the specialized endothelial cells that give rise to HSCs, to the perivascular niche endothelial cells that regulate HSC homeostasis. Recent studies have dissected the unique molecular and physical properties of the endothelial cells in the HSC vascular niche and their role in HSC biology, which may be manipulated to enhance hematopoietic stem cell transplantation therapies.

Circulating endothelial cells as marker of endothelial damage in male hypogonadism.

Science.gov (United States)

Milardi, Domenico; Grande, Giuseppe; Giampietro, Antonella; Vendittelli, Francesca; Palumbo, Sara; Tartaglione, Linda; Marana, Riccardo; Pontecorvi, Alfredo; de Marinis, Laura; Zuppi, Cecilia; Capoluongo, Ettore

2012-01-01

Testosterone deficiency has become a frequently diagnosed condition in today's society affected by epidemic obesity, and is associated with cardiovascular risk. Recent studies have established the importance of altered vascular endothelium function in cardiovascular disease. The damage to the endothelium might also cause endothelial cell detachment, resulting in increased numbers of circulating endothelial cells (CEC) within the bloodstream. To evaluate whether hypogonadism could modify CEC count in peripheral bloodstream, we investigated peripheral blood CEC count using the CellSearch System, a semiautomatic method to accurately and reliably enumerate CECs, which are sorted based on a CD146(+), CD105(+), DAPI(+), CD45(-) phenotype, in a population of 20 patients with hypogonadism. The control group comprised 10 age- and sex-matched healthy participants. CEC count per milliliter was significantly increased in patients with hypogonadism vs the control group. In the group with hypogonadism, an inverse exponential correlation was present between testosterone levels and CEC count per milliliter. A direct linear correlation was present between waist circumference and CECs and between body mass index and CECs. The regression analysis showed that testosterone was the significant independent determinant of CECs. Our results underline that male hypogonadism is associated with endothelial dysfunction. The correlation between CEC and waist circumference underlines that visceral obesity may be synergically implicated in this regulation. Future studies are required to unveil the mechanisms involved in the pathogenesis of testosterone-induced endothelial disfunction, which may provide novel therapeutic targets to be incorporated in the management of hypogonadism.

A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.

Science.gov (United States)

Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun

2018-07-01

Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

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Jason Tran

Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

DEFF Research Database (Denmark)

Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

2018-01-01

PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

Direct conversion of injury-site myeloid cells to fibroblast-like cells of granulation tissue.

Science.gov (United States)

Sinha, Mithun; Sen, Chandan K; Singh, Kanhaiya; Das, Amitava; Ghatak, Subhadip; Rhea, Brian; Blackstone, Britani; Powell, Heather M; Khanna, Savita; Roy, Sashwati

2018-03-05

Inflammation, following injury, induces cellular plasticity as an inherent component of physiological tissue repair. The dominant fate of wound macrophages is unclear and debated. Here we show that two-thirds of all granulation tissue fibroblasts, otherwise known to be of mesenchymal origin, are derived from myeloid cells which are likely to be wound macrophages. Conversion of myeloid to fibroblast-like cells is impaired in diabetic wounds. In cross-talk between keratinocytes and myeloid cells, miR-21 packaged in extracellular vesicles (EV) is required for cell conversion. EV from wound fluid of healing chronic wound patients is rich in miR-21 and causes cell conversion more effectively compared to that by fluid from non-healing patients. Impaired conversion in diabetic wound tissue is rescued by targeted nanoparticle-based delivery of miR-21 to macrophages. This work introduces a paradigm wherein myeloid cells are recognized as a major source of fibroblast-like cells in the granulation tissue.

Plasma levels of stromal cell-derived factor-1 (CXCL12) and circulating endothelial progenitor cells in women with idiopathic heavy menstrual bleeding.

Science.gov (United States)

Elsheikh, E; Andersson, E; Sylvén, C; Ericzon, B-G; Palmblad, J; Mints, M

2014-01-01

Do plasma levels of stromal cell-derived factor-1 (CXCL12, sometimes termed SDF-1) and the numbers of circulating endothelial progenitor cells (EPCs), EPC colony-forming units (EPC-CFU) and mature endothelial cells (ECs) differ between women with idiopathic heavy menstrual bleeding of endometrial origin (HMB-E) and controls and are they related to plasma levels of other angiogenic growth factors? Angiogenesis is altered in women with HMB-E, characterized by a reduction in mean plasma levels of CXCL12, a low number of EPCs-CFUs and a high level of circulating ECs. Plasma levels of CXCL12 are significantly higher during the proliferative than the secretory phase of the menstrual cycle in healthy women and exhibit a negative correlation with blood EPC-CFUs. A prospective cohort study in a university hospital setting. Between 2008 and 2009 10 HMB-E patients were recruited from Karolinska University Hospital. Ten healthy women were also included in the analysis. Ten healthy control women and 10 HMB-E patients, all with regular menstrual cycles, provided 4 blood samples during a single menstrual cycle: 2 in the proliferative phase, 1 at ovulation and 1 in the secretory phase. We assessed plasma levels of CXCL12, vascular endothelial growth factor A(165) (VEGFA), basic fibroblast growth factor (bFGF) and granulocyte and granulocyte-macrophage colony-stimulating factors by ELISA. We counted circulating EPC-CFUs by culture, and ECs and EPCs by flow cytometry and immunostaining for cell surface markers. Plasma levels of CXCL12 were significantly lower in HMB-E patients compared with control women (P Market Insurance. The authors have no conflict of interest to declare.

Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

Science.gov (United States)

Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

2014-10-14

Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

Lymphocyte adhesion to endothelial cell (EC) is stimulated by phorbol esters

International Nuclear Information System (INIS)

Haskard, D.; Cavender, D.; Ziff, M.

1986-01-01

The effect of phorbol esters on T cell adhesion to EC has been studied. The phorbol esters 12-0-tetradecanoyl-phorbol-13-acetate and 4-beta-phorbol-12-13-dibutyrate, but not the biologically inert 4-0-methyl-phorbol-12-13-didecanoate strongly increased the binding of 51 Cr-labeled T cells to human umbilical vein EC monolayers in microtiter wells. Increase in binding was observed at 0.3 ng/ml with maximal enhancement at 50 ng/ml. Both unstimulated and phorbol ester activated T cells displayed a substantially greater binding affinity for EC than for fibroblasts or plastic. Binding enhancement occurred within one minute, with maximal increase after 15 min. Preincubation studies showed that binding enhancement was entirely attributable to an effect on T cells, with no action on EC. Additive binding enhancement was seen when phorbol esters and reagents that increase adhesion by actions on EC (LPS, IL-1 and IFN-γ) were used together. Increase in adhesion of activated T lymphocytes to EC may explain the greater emigration of activated T cells than small resting T cells into inflammatory foci in vivo. The rapid onset of the phorbol effect suggests that this may be an important mechanism for immediate localization of circulating T cells in the cellular immune response, activated, perhaps, at the endothelial blood-tissue interface

Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

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Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

2004-07-14

Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

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Shuei-Kuen Tseng

2014-09-01

Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

International Nuclear Information System (INIS)

Rubin, D.B.; Drab, E.A.; Ward, W.F.; Bauer, K.D.

1988-01-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and an increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous [3H]thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro

MicroRNAs in Hyperglycemia Induced Endothelial Cell Dysfunction

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Maskomani Silambarasan

2016-04-01

Full Text Available Hyperglycemia is closely associated with prediabetes and Type 2 Diabetes Mellitus. Hyperglycemia increases the risk of vascular complications such as diabetic retinopathy, diabetic nephropathy, peripheral vascular disease and cerebro/cardiovascular diseases. Under hyperglycemic conditions, the endothelial cells become dysfunctional. In this study, we investigated the miRNA expression changes in human umbilical vein endothelial cells exposed to different glucose concentrations (5, 10, 25 and 40 mM glucose and at various time intervals (6, 12, 24 and 48 h. miRNA microarray analyses showed that there is a correlation between hyperglycemia induced endothelial dysfunction and miRNA expression. In silico pathways analyses on the altered miRNA expression showed that the majority of the affected biological pathways appeared to be associated to endothelial cell dysfunction and apoptosis. We found the expression of ten miRNAs (miR-26a-5p, -26b-5p, 29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -140-5p, -192-5p, -221-3p and -320a to increase gradually with increasing concentration of glucose. These miRNAs were also found to be involved in endothelial dysfunction. At least seven of them, miR-29b-3p, -29c-3p, -125b-1-3p, -130b-3p, -221-3p, -320a and -192-5p, can be correlated to endothelial cell apoptosis.

Soluble CD54 induces human endothelial cells ex vivo expansion useful for cardiovascular regeneration and tissue engineering application

KAUST Repository

Malara, N.M.

2015-03-01

Aim: Consistent expansion of primary human endothelial cells in vitro is critical in the development of engineered tissue. A variety of complex culture media and techniques developed from different basal media have been reported with alternate success. Incongruous results are further confounded by donor-to-donor variability and cellular source of derivation. Our results demonstrate how to overcome these limitations using soluble CD54 (sCD54) as additive to conventional culture medium. Methods and results: Isolated primary fragment of different vessel types was expanded in Ham\\'s F12 DMEM, enriched with growth factors, Fetal Calf Serum and conditioned medium of Human Umbilical Vein Endothelial Cells (HUVEC) collected at different passages. Cytokine content of culture media was analyzed in order to identify the soluble factors correlating with better proliferation profile. sCD54 was found to induce the in vitro expansion of human endothelial cells (HECs) independently from the vessels source and even in the absence of HUVEC-conditioned medium. The HECs cultivated in the presence of sCD54 (50 ng/ml), resulted positive for the expression of CD146 and negative for CD45, and lower fibroblast contamination. Cells were capable to proliferate with an S phase of 25%, to produce vascular endothelial growth factor, VEGF, (10 ng/ml) and to give origin to vessel-like tubule in vitro. Conclusion: Our results demonstrate that sCD54 is an essential factor for the in-vitro expansion of HECs without donor and vessel-source variability. Resulting primary cultures can be useful, for tissue engineering in regenerative medicine (e.g. artificial micro tissue generation, coating artificial heart valve etc.) and bio-nanotechnology applications. © 2015 The Authors. Published by Elsevier Ireland Ltd.

Cell-free assay measuring repair DNA synthesis in human fibroblasts

International Nuclear Information System (INIS)

Ciarrocchi, G.; Linn, S.

1978-01-01

Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

Inhibition of microparticle release triggers endothelial cell apoptosis and detachment

NARCIS (Netherlands)

Abid Hussein, Mohammed N.; Böing, Anita N.; Sturk, Augueste; Hau, Chi M.; Nieuwland, Rienk

2007-01-01

Endothelial cell cultures contain caspase 3-containing microparticles (EMP), which are reported to form during or after cell detachment. We hypothesize that also adherent endothelial cells release EMP, thus protecting these cells from caspase 3 accumulation, detachment and apoptosis. Human umbilical

Bee products prevent VEGF-induced angiogenesis in human umbilical vein endothelial cells

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Mishima Satoshi

2009-11-01

Full Text Available Abstract Background Vascular endothelial growth factor (VEGF is a key regulator of pathogenic angiogenesis in diseases such as cancer and diabetic retinopathy. Bee products [royal jelly (RJ, bee pollen, and Chinese red propolis] from the honeybee, Apis mellifera, have been used as traditional health foods for centuries. The aim of this study was to investigate the anti-angiogenic effects of bee products using human umbilical vein endothelial cells (HUVECs. Methods In an in vitro tube formation assay, HUVECs and fibroblast cells were incubated for 14 days with VEGF and various concentrations of bee products [RJ, ethanol extract of bee pollen, ethanol extract of Chinese red propolis and its constituent, caffeic acid phenethyl ester (CAPE]. To clarify the mechanism of in vitro angiogenesis, HUVEC proliferation and migration were induced by VEGF with or without various concentrations of RJ, bee pollen, Chinese red propolis, and CAPE. Results RJ, bee pollen, Chinese red propolis, and CAPE significantly suppressed VEGF-induced in vitro tube formation in the descending order: CAPE > Chinese red propolis >> bee pollen > RJ. RJ and Chinese red propolis suppressed both VEGF-induced HUVEC proliferation and migration. In contrast, bee pollen and CAPE suppressed only the proliferation. Conclusion Among the bee products, Chinese red propolis and CAPE in particular showed strong suppressive effects against VEGF-induced angiogenesis. These findings indicate that Chinese red propolis and CAPE may have potential as preventive and therapeutic agents against angiogenesis-related human diseases.

Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

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Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

2017-11-01

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

Effects of blood products on inflammatory response in endothelial cells in vitro.

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Martin Urner

Full Text Available BACKGROUND: Transfusing blood products may induce inflammatory reactions within the vascular compartment potentially leading to a systemic inflammatory response. Experiments were designed to assess the inflammatory potential of different blood products in an endothelial cell-based in vitro model and to compare baseline levels of potentially activating substances in transfusion products. METHODS: The inflammatory response from pre-activated (endotoxin-stimulated and non-activated endothelial cells as well as neutrophil endothelial transmigration in response to packed red blood cells (PRBC, platelet concentrates (PC and fresh frozen plasma (FFP was determined. Baseline inflammatory mediator and lipid concentrations in blood products were evaluated. RESULTS: Following incubation with all blood products, an increased inflammatory mediator release from endothelial cells was observed. Platelet concentrates, and to a lesser extent also FFP, caused the most pronounced response, which was accentuated in already pre-stimulated endothelial cells. Inflammatory response of endothelial cells as well as blood product-induced migration of neutrophils through the endothelium was in good agreement with the lipid content of the according blood product. CONCLUSION: Within the group of different blood transfusion products both PC and FFP have a high inflammatory potential with regard to activation of endothelial cells. Inflammation upon blood product exposure is strongly accentuated when endothelial cells are pre-injured. High lipid contents in the respective blood products goes along with an accentuated inflammatory reaction from endothelial cells.

Infections and endothelial cells

NARCIS (Netherlands)

Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

2003-01-01

Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

International Nuclear Information System (INIS)

Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

2014-01-01

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

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Fuchigami, Takao [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kibe, Toshiro [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Nishizawa, Yoshiaki [Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Hijioka, Hiroshi; Fujii, Tomomi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ueda, Masahiro [Natural Science Centre for Research and Education, Kagoshima University, 1-21-24 Koorimoto, Kagoshima 890-8580 (Japan); Nakamura, Norifumi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kiyono, Tohru [Department of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuouku, Tokyo 104-0045 (Japan); Kishida, Michiko, E-mail: kmichiko@m2.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

2014-09-05

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

Endothelial cell energy metabolism, proliferation, and apoptosis in pulmonary hypertension.

Science.gov (United States)

Xu, Weiling; Erzurum, Serpil C

2011-01-01

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary hemodynamics and excessive growth and dysfunction of the endothelial cells that line the arteries in PAH lungs. Establishment of methods for culture of pulmonary artery endothelial cells from PAH lungs has provided the groundwork for mechanistic translational studies that confirm and extend findings from model systems and spontaneous pulmonary hypertension in animals. Endothelial cell hyperproliferation, survival, and alterations of biochemical-metabolic pathways are the unifying endothelial pathobiology of the disease. The hyperproliferative and apoptosis-resistant phenotype of PAH endothelial cells is dependent upon the activation of signal transducer and activator of transcription (STAT) 3, a fundamental regulator of cell survival and angiogenesis. Animal models of PAH, patients with PAH, and human PAH endothelial cells produce low nitric oxide (NO). In association with the low level of NO, endothelial cells have reduced mitochondrial numbers and cellular respiration, which is associated with more than a threefold increase in glycolysis for energy production. The shift to glycolysis is related to low levels of NO and likely to the pathologic expression of the prosurvival and proangiogenic signal transducer, hypoxia-inducible factor (HIF)-1, and the reduced mitochondrial antioxidant manganese superoxide dismutase (MnSOD). In this article, we review the phenotypic changes of the endothelium in PAH and the biochemical mechanisms accounting for the proliferative, glycolytic, and strongly proangiogenic phenotype of these dysfunctional cells, which consequently foster the panvascular progressive pulmonary remodeling in PAH. © 2011 American Physiological Society.

Tumor endothelial marker 5 expression in endothelial cells during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of cell proliferation

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Vallon, Mario, E-mail: m.vallon@arcor.de [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany); Rohde, Franziska; Janssen, Klaus-Peter [Chirurgische Klinik und Poliklinik, Technische Universitaet Muenchen, Munich (Germany); Essler, Markus [Nuklearmedizinische Klinik und Poliklinik, Technische Universitaet Muenchen, Ismaninger Strasse 22, 81675 Munich (Germany)

2010-02-01

Tumor endothelial marker (TEM) 5 is an adhesion G-protein-coupled receptor upregulated in endothelial cells during tumor and physiologic angiogenesis. So far, the mechanisms leading to upregulation of TEM5 and its function during angiogenesis have not been identified. Here, we report that TEM5 expression in endothelial cells is induced during capillary-like network formation on Matrigel, during capillary morphogenesis in a three-dimensional collagen I matrix, and upon confluence on a two-dimensional matrix. TEM5 expression was not induced by a variety of soluble angiogenic factors, including VEGF and bFGF, in subconfluent endothelial cells. TEM5 upregulation was blocked by toxin B from Clostridium difficile, an inhibitor of the small GTPases Rho, Rac, and Cdc42. The Rho inhibitor C3 transferase from Clostridium botulinum did not affect TEM5 expression, whereas the Rac inhibitor NSC23766 suppressed TEM5 upregulation. An excess of the soluble TEM5 extracellular domain or an inhibitory monoclonal TEM5 antibody blocked contact inhibition of endothelial cell proliferation resulting in multilayered islands within the endothelial monolayer and increased vessel density during capillary formation. Based on our results we conclude that TEM5 expression during capillary morphogenesis is induced by the small GTPase Rac and mediates contact inhibition of proliferation in endothelial cells.

Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR1 activation

International Nuclear Information System (INIS)

Blanc-Brude, Olivier P.; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

2005-01-01

Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR 1 ). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR 1 -deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR 1 -specific agonists and inhibitors were used to demonstrate that PAR 1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR 1 and not PAR 2 . These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis

Transcellular transport of cobalamin in aortic endothelial cells.

Science.gov (United States)

Hannibal, Luciana; Bolisetty, Keerthana; Axhemi, Armend; DiBello, Patricia M; Quadros, Edward V; Fedosov, Sergey; Jacobsen, Donald W

2018-05-09

Cobalamin [Cbl (or B 12 )] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B 12 , namely, the complex formed between serum transcobalamin and B 12 , designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [ 57 Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [ 57 Co]-CNCbl, and via transcytosis as [ 57 Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [ 57 Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various β-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ∼40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B 12 via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B 12 .-Hannibal, L., Bolisetty, K., Axhemi, A., DiBello, P

Sphingosine 1-Phosphate Induces Platelet/Endothelial Cell Adhesion Molecule-1 Tyrosine Phosphorylation in Bovine Aortic Endothelial Cells through a PP2-Inhibitable Mechanism

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Yu-Ting Huang

2007-12-01

Full Text Available Sphingosine-1-phosphate (S1P is a low-molecular-weight phospholipid derivative released by activated platelets. S1P transduces signals through a family of G protein-coupled receptors to modulate various physiological behaviors of endothelial cells. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31 is a 130-kDa protein expressed on the surfaces of leukocytes, platelets, and endothelial cells. Upon PECAM-1 activation, its cytoplasmic tyrosine residues become phosphorylated and bind with SH2 domain-containing proteins, thus leading to the downstream functions mediated by PECAM-1. In the present study, we found that S1P induced PECAM-1 tyrosine phosphorylation and SHP-2 association in bovine aortic endothelial cells (BAECs by immunoprecipitation and western blotting. The pretreatment of BAECs with a series of chemical inhibitors to determine the signaling pathway showed that the PECAM-1 phosphorylation was inhibited by PP2, indicating the participation of Src family kinases. These results demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in BAECs through mediation of Src family kinases, and this may regulate the physiological behaviors of endothelial cells.

A benzoxazine derivative induces vascular endothelial cell apoptosis in the presence of fibroblast growth factor-2 by elevating NADPH oxidase activity and reactive oxygen species levels.

Science.gov (United States)

Zhao, Jing; He, Qiuxia; Cheng, Yizhe; Zhao, Baoxiang; Zhang, Yun; Zhang, Shangli; Miao, Junying

2009-09-01

Previously, we found that 6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine (DBO) promoted apoptosis of human umbilical vascular endothelial cells (HUVECs) deprived of growth factors. In this study, we aimed to investigate the effect of DBO and its mechanism of action on angiogenesis and apoptosis of HUVECs in the presence of fibroblast growth factor-2 (FGF-2), which promotes angiogenesis and inhibits apoptosis in vivo and in vitro. DBO significantly inhibited capillary-like tube formation by promoting apoptosis of HUVECs in the presence of FGF-2 in vitro. Furthermore, DBO elevated the levels of reactive oxygen species (ROS) and nitric oxide (NO) and increased the activity of NADPH oxidase and inducible nitric oxide synthase (iNOS) in promoting apoptosis under this condition. Moreover, when NADPH oxidase was inhibited by its specific inhibitor, dibenziodolium chloride (DPI), DBO could not elevate ROS and NO levels in HUVECs. The data suggest that DBO is a new modulator of apoptosis in vitro, and it might function by increasing the activity of NADPH oxidase and iNOS, subsequently elevating the levels of ROS and NO in HUVECs. The findings of this study provide a new small molecule for investigating the FGF-2/NADPH oxidase/iNOS signaling pathway in apoptosis.

A novel minimally-invasive method to sample human endothelial cells for molecular profiling.

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Stephen W Waldo

Full Text Available The endothelium is a key mediator of vascular homeostasis and cardiovascular health. Molecular research on the human endothelium may provide insight into the mechanisms underlying cardiovascular disease. Prior methodology used to isolate human endothelial cells has suffered from poor yields and contamination with other cell types. We thus sought to develop a minimally invasive technique to obtain endothelial cells derived from human subjects with higher yields and purity.Nine healthy volunteers underwent endothelial cell harvesting from antecubital veins using guidewires. Fluorescence-activated cell sorting (FACS was subsequently used to purify endothelial cells from contaminating cells using endothelial surface markers (CD34/CD105/CD146 with the concomitant absence of leukocyte and platelet specific markers (CD11b/CD45. Endothelial lineage in the purified cell population was confirmed by expression of endothelial specific genes and microRNA using quantitative polymerase chain reaction (PCR.A median of 4,212 (IQR: 2161-6583 endothelial cells were isolated from each subject. Quantitative PCR demonstrated higher expression of von Willebrand Factor (vWF, P<0.001, nitric oxide synthase 3 (NOS3, P<0.001 and vascular cell adhesion molecule 1 (VCAM-1, P<0.003 in the endothelial population compared to similarly isolated leukocytes. Similarly, the level of endothelial specific microRNA-126 was higher in the purified endothelial cells (P<0.001.This state-of-the-art technique isolates human endothelial cells for molecular analysis in higher purity and greater numbers than previously possible. This approach will expedite research on the molecular mechanisms of human cardiovascular disease, elucidating its pathophysiology and potential therapeutic targets.

Low-Intensity Pulsed Ultrasound Prevents the Oxidative Stress Induced Endothelial-Mesenchymal Transition in Human Aortic Endothelial Cells

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Jiamin Li

2018-02-01

Full Text Available Background/Aims: Endothelial-mesenchymal transition (EndMT has been shown to take part in the generation and progression of diverse diseases, involving a series of changes leading to a loss of their endothelial characteristics and an acquirement of properties typical of mesenchymal cells. Low-intensity pulsed ultrasound (LIPUS is a new therapeutic option that has been successfully used in fracture healing. However, whether LIPUS can inhibit oxidative stress-induced endothelial cell damages through inhibiting EndMT remained unknown. This study aimed to investigate the protective effects of LIPUS against oxidative stress-induced endothelial cell damages and the underlying mechanisms. Methods: EndMT was induced by H2O2 (100 µm for seven days. Human aortic endothelial cells (HAECs were exposed to H2O2 with or without LIPUS treatment for seven days. The expression of EndMT markers (CD31, VE-cadherin, FSP1 and α-SMA were analyzed. The levels of total and phosphorylated PI3K and AKT proteins were detected by Western Blot analysis. Cell chemotaxis was determined by wound healing and transwell assay. Results: LIPUS relieved EndMT by decreasing ROS accumulation and increasing activation of the PI3K signaling cascade. LIPUS alleviated the migration of EndMT-derived mesenchymal-like cells through reducing extracellular matrix (ECM deposition that is associated with matrix metallopeptidase (MMP proteolytic activity and collagen production. Conclusion: LIPUS produces cytoprotective effects against oxidative injuries to endothelial cells through suppressing the oxidative stress-induced EndMT, activating the PI3K/AKT pathway under oxidative stress, and limiting cell migration and excessive ECM deposition.

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

Science.gov (United States)

Alghanem, Ahmad F; Wilkinson, Emma L; Emmett, Maxine S; Aljasir, Mohammad A; Holmes, Katherine; Rothermel, Beverley A; Simms, Victoria A; Heath, Victoria L; Cross, Michael J

2017-08-01

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells

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Kobayashi, Hideki; Butler, Jason M.; O'Donnell, Rebekah; Kobayashi, Mariko; Ding, Bi-Sen; Bonner, Bryant; Chiu, Vi K.; Nolan, Daniel J.; Shido, Koji; Benjamin, Laura; Rafii, Shahin

2010-01-01

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34−Flt3− KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34−Flt3− KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt–mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs. PMID:20972423

Effect of tributyltin on mammalian endothelial cell integrity.

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Botelho, G; Bernardini, C; Zannoni, A; Ventrella, V; Bacci, M L; Forni, M

2015-01-01

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

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Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

International Nuclear Information System (INIS)

Cogan, Nicola; Baird, Duncan M.; Phillips, Ryan; Crompton, Lucy A.; Caldwell, Maeve A.; Rubio, Miguel A.; Newson, Roger; Lyng, Fiona; Case, C. Patrick

2010-01-01

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

Energy Technology Data Exchange (ETDEWEB)

Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

2010-01-05

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

Proliferating fibroblasts and HeLa cells co-cultured in vitro reciprocally influence growth patterns, protein expression, chromatin features and cell survival.

Science.gov (United States)

Delinasios, John G; Angeli, Flora; Koumakis, George; Kumar, Shant; Kang, Wen-Hui; Sica, Gigliola; Iacopino, Fortunata; Lama, Gina; Lamprecht, Sergio; Sigal-Batikoff, Ina; Tsangaris, George T; Farfarelos, Christos D; Farfarelos, Maria C; Vairaktaris, Eleftherios; Vassiliou, Stavros; Delinasios, George J

2015-04-01

to identify biological interactions between proliferating fibroblasts and HeLa cells in vitro. Fibroblasts were isolated from both normal and tumour human tissues. Coverslip co-cultures of HeLa and fibroblasts in various ratios with medium replacement every 48 h were studied using fixed cell staining with dyes such as Giemsa and silver staining, with immunochemistry for Ki-67 and E-cadherin, with dihydrofolate reductase (DHFR) enzyme reaction, as well as live cell staining for non-specific esterases and lipids. Other techniques included carmine cell labeling, autoradiography and apoptosis assessment. Under conditions of feeding and cell: cell ratios allowing parallel growth of human fibroblasts and HeLa cells, co-cultured for up to 20 days, a series of phenomena occur consecutively: profound affinity between the two cell types and exchange of small molecules; encircling of the HeLa colonies by the fibroblasts and enhanced growth of both cell types at their contact areas; expression of carbonic anhydrase in both cell types and high expression of non-specific esterases and cytoplasmic argyrophilia in the surrounding fibroblasts; intense production and secretion of lipid droplets by the surrounding fibroblasts; development of a complex net of argyrophilic projections of the fibroblasts; E-cadherin expression in the HeLa cells; from the 10th day onwards, an increasing detachment of batches of HeLa cells at the peripheries of colonies and appearance of areas with many multi-nucleated and apoptotic HeLa cells, and small HeLa fragments; from the 17th day, appearance of fibroblasts blocked at the G2-M phase. Co-cultures at approximately 17-20 days display a cell-cell fight with foci of (a) sparse growth of both cell types, (b) overgrowth of the fibroblasts and (c) regrowth of HeLa in small colonies. These results indicate that during their interaction with HeLa cells in vitro, proliferating fibroblasts can be activated against HeLa. This type of activation is not observed

Endothelial cell cultures as a tool in biomaterial research

NARCIS (Netherlands)

Kirkpatrick, CJ; Otto, M; van Kooten, T; Krump, [No Value; Kriegsmann, J; Bittinger, F

1999-01-01

Progress in biocompatibility and tissue engineering would today be inconceivable without the aid of in vitro techniques. Endothelial cell cultures represent a valuable tool not just in haemocompatibility testing, but also in the concept of designing hybrid organs. In the past endothelial cells (EC)

Melanoma Cells Can Adopt the Phenotype of Stromal Fibroblasts and Macrophages by Spontaneous Cell Fusion in Vitro.

Science.gov (United States)

Kemény, Lajos V; Kurgyis, Zsuzsanna; Buknicz, Tünde; Groma, Gergely; Jakab, Ádám; Zänker, Kurt; Dittmar, Thomas; Kemény, Lajos; Németh, István B

2016-06-02

After the removal of primary cutaneous melanoma some patients develop local recurrences, even after having histologically tumor-free re-excision. A potential explanation behind this phenomenon is that tumor cells switch their phenotype, making their recognition via standard histopathological assessments extremely difficult. Tumor-stromal cell fusion has been proposed as a potential mechanism for tumor cells to acquire mesenchymal traits; therefore, we hypothesized that melanoma cells could acquire fibroblast- and macrophage-like phenotypes via cell fusion. We show that melanoma cells spontaneously fuse with human dermal fibroblasts and human peripheral blood monocytes in vitro. The hybrid cells' nuclei contain chromosomes from both parental cells and are indistinguishable from the parental fibroblasts or macrophages based on their morphology and immunophenotype, as they could lose the melanoma specific MART1 marker, but express the fibroblast marker smooth muscle actin or the macrophage marker CD68. Our results suggest that, by spontaneous cell fusion in vitro, tumor cells can adopt the morphology and immunophenotype of stromal cells while still carrying oncogenic, tumor-derived genetic information. Therefore, melanoma-stromal cell fusion might play a role in missing tumor cells by routine histopathological assessments.

Apoptosis of Endothelial Cells by 13-HPODE Contributes to Impairment of Endothelial Barrier Integrity

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Valerie E. Ryman

2016-01-01

Full Text Available Inflammation is an essential host response during bacterial infections such as bovine mastitis. Endothelial cells are critical for an appropriate inflammatory response and loss of vascular barrier integrity is implicated in the pathogenesis of Streptococcus uberis-induced mastitis. Previous studies suggested that accumulation of linoleic acid (LA oxygenation products derived from 15-lipoxygenase-1 (15-LOX-1 metabolism could regulate vascular functions. The initial LA derivative from the 15-LOX-1 pathway, 13-hydroperoxyoctadecadienoic acid (HPODE, can induce endothelial death, whereas the reduced hydroxyl product, 13-hydroxyoctadecadienoic acid (HODE, is abundantly produced during vascular activation. However, the relative contribution of specific LA-derived metabolites on impairment of mammary endothelial integrity is unknown. Our hypothesis was that S. uberis-induced LA-derived 15-LOX-1 oxygenation products impair mammary endothelial barrier integrity by apoptosis. Exposure of bovine mammary endothelial cells (BMEC to S. uberis did not increase 15-LOX-1 LA metabolism. However, S. uberis challenge of bovine monocytes demonstrated that monocytes may be a significant source of both 13-HPODE and 13-HODE during mastitis. Exposure of BMEC to 13-HPODE, but not 13-HODE, significantly reduced endothelial barrier integrity and increased apoptosis. Changing oxidant status by coexposure to an antioxidant during 13-HPODE treatment prevented adverse effects of 13-HPODE, including amelioration of apoptosis. A better understanding of how the oxidant status of the vascular microenvironment impacts endothelial barrier properties could lead to more efficacious treatments for S. uberis mastitis.

Changes of junctions of endothelial cells in coronary sclerosis: A review

Directory of Open Access Journals (Sweden)

Li-Zi Zhang

2016-03-01

Full Text Available Atherosclerosis, the major cause of cardiovascular diseases, has been a leading contributor to morbidity and mortality in the United States and it has been on the rise globally. Endothelial cell–cell junctions are critical for vascular integrity and maintenance of vascular function. Endothelial cell junctions dysfunction is the onset step of future coronary events and coronary artery disease. Keywords: Coronary atherosclerosis, Junctions, Endothelial cells

Young endothelial cells revive aging blood.

Science.gov (United States)

Chang, Vivian Y; Termini, Christina M; Chute, John P

2017-11-01

The hematopoietic system declines with age, resulting in decreased hematopoietic stem cell (HSC) self-renewal capacity, myeloid skewing, and immune cell depletion. Aging of the hematopoietic system is associated with an increased incidence of myeloid malignancies and a decline in adaptive immunity. Therefore, strategies to rejuvenate the hematopoietic system have important clinical implications. In this issue of the JCI, Poulos and colleagues demonstrate that infusions of bone marrow (BM) endothelial cells (ECs) from young mice promoted HSC self-renewal and restored immune cell content in aged mice. Additionally, delivery of young BM ECs along with HSCs following total body irradiation improved HSC engraftment and enhanced survival. These results suggest an important role for BM endothelial cells (ECs) in regulating hematopoietic aging and support further research to identify the rejuvenating factors elaborated by BM ECs that restore HSC function and the immune repertoire in aged mice.

Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

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Kristin Dawson

Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

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Jason A Neidleman

2017-02-01

Full Text Available Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

Science.gov (United States)

Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

Characterization of vascular endothelial progenitor cells from chicken bone marrow

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Bai Chunyu

2012-05-01

Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

Drug-induced in vitro inhibition of neutrophil-endothelial cell adhesion.

Science.gov (United States)

Pellegatta, F.; Lu, Y.; Radaelli, A.; Zocchi, M. R.; Ferrero, E.; Chierchia, S.; Gaja, G.; Ferrero, M. E.

1996-01-01

1. Leukocyte-endothelial cell interactions play an important role during ischaemia-reperfusion events. Adhesion molecules are specifically implicated in this interaction process. 2. Since defibrotide has been shown to be an efficient drug in reducing damage due to ischaemia-reperfusion in many experimental models, we analysed the effect of defibrotide in vitro on leukocyte adhesion to endothelial cells in basal conditions and after their stimulation. 3. In basal conditions, defibrotide (1000 micrograms ml-1) partially inhibited leukocyte adhesion to endothelial cells by 17.3% +/- 3.6 (P defibrotide. 5. This result was confirmed in NIH/3T3-ICAM-1 transfected cells. 6. We conclude that defibrotide is able to interfere with leukocyte adhesion to endothelial cells mainly in activated conditions and that the ICAM-1/LFA-1 adhesion system is involved in the defibrotide mechanism of action. PMID:8762067

Catalase and superoxide dismutase conjugated with platelet-endothelial cell adhesion molecule antibody distinctly alleviate abnormal endothelial permeability caused by exogenous reactive oxygen species and vascular endothelial growth factor.

Science.gov (United States)

Han, Jingyan; Shuvaev, Vladimir V; Muzykantov, Vladimir R

2011-07-01

Reactive oxygen species (ROS) superoxide anion (O(2)()) and hydrogen peroxide (H(2)O(2)) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H(2)O(2)-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H(2)O(2) in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O(2)() in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of

Calmodulin-Dependent Protein Kinase mediates Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

Science.gov (United States)

Love, Felisha D.; Melhado, Caroline; Bosah, Francis; Harris-Hooker, Sandra A.; Sanford, Gary L.

1997-01-01

A number of basic cellular functions, e.g., electrolyte concentration cell growth rate, glucose utilization, bone formation, response to growth stimulation and exocytosis are modified by microgravity or during spaceflight. Studies with intact animal during spaceflights have found lipid accumulations within the lumen of the vasculature and degeneration of the vascular wall. Capillary alterations with extensive endothelial invaginations were also seen. Hemodynamic studies have shown that there is a redistribution of blood from the lower extremities to the upper part of the body; this will alter vascular permeability, resulting in leakage into surrounding tissues. These studies indicate that changes in gravity will affect a number of physiological systems, including the vasculature. However, few studies have addressed the effect of microgravity on vascular cell function and metabolism. A major problem with ground based studies is that achieving a true microgravity hand, environment for prolonged period is not possible. On the other increasing gravity (i.e., hypergravity) is easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell limes (e.g., chick embryo fibroblasts) while decreasing cell motility and slowing liver regeneration following partial hepatectomy. These studies suggest that hypergravity will alter the behavior of most cells. Several investigators have shown that hypergravity affects the expression of the early response genes (c-fos and c-myc) and the activation of several protein kinases (PK's) in cells (10,11). In this study we investigated whether hypergravity alters the expression of f-actin by aortic endothelial cells, and the possible role of protein kinases (calmodulin(II)-dependent and PKA) as mediators of these effects.

The Bony Side of Endothelial Cells in Prostate Cancer.

Science.gov (United States)

Peng, Jia; Kang, Yibin

2017-06-05

Prostate cancer bone metastases are primarily osteoblastic, but the source of bone-forming cells in these lesions remains poorly defined. In this issue of Developmental Cell, Lin et al. (2017) demonstrate that tumor-associated endothelial cells can give rise to osteoblasts in prostate cancer through endothelial-to-osteoblast (EC-to-OSB) conversion. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of sunitinib combined with ionizing radiation on endothelial cells

International Nuclear Information System (INIS)

Zhang Haiping; Jiao Xiaodong; Li Rui; Wang Jiejun; Takayama, Koichi; Su Bo

2011-01-01

The aims of present study were to evaluate the efficacy of combining sunitinib with ionizing radiation (IR) on endothelial cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were exposed to IR with or without sunitinib pretreatment. Apoptosis assay and cell cycle distribution were analyzed by flow cytometry. Clonogenic survival assay at 3 Gy dose with or without sunitinib was performed. The activity of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway was detected by Western immunoblot. Lewis lung carcinoma mouse model was built to examine the effect of combination therapy on endothelial cells in vivo. Microvasculature changes were detected by immunohistochemistry using anti-CD31 antibody. Our results showed combination therapy of sunitinib and IR significantly increased apoptosis of endothelial cells and inhibited colony formation compared to sunitinib or radiotherapy alone. It also resulted in cell cycle redistribution (decreasing cells in S phase and increasing cells in G2/M phase). The activity of PI3K/Akt signal pathway was inhibited, which could be the potential mechanisms that account for the enhanced radiation response induced by sunitinib. In vivo analysis showed that combination therapy significantly decreased microvasculature formation. The results demonstrated that combination therapy of sunitinib and IR has the potential to increase the cytotoxic effects on endothelial cells. (author)

Apelin is a novel angiogenic factor in retinal endothelial cells

International Nuclear Information System (INIS)

Kasai, Atsushi; Shintani, Norihito; Oda, Maki; Kakuda, Michiya; Hashimoto, Hitoshi; Matsuda, Toshio; Hinuma, Shuji; Baba, Akemichi

2004-01-01

There has been much focus recently on the possible functions of apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, in cardiovascular and central nervous systems. We report a new function of apelin as a novel angiogenic factor in retinal endothelial cells. The retinal endothelial cell line RF/6A highly expressed both apelin and APJ transcripts, while human umbilical venous endothelial cells (HUVECs) only expressed apelin mRNA. In accordance with these observations, apelin at concentrations of 1 pM-1 μM significantly enhanced migration, proliferation, and capillary-like tube formation of RF/6A cells, but not those of HUVECs, whereas VEGF stimulates those parameters of both cell types. In vivo Matrigel plug assay for angiogenesis, the inclusion of 1 nM apelin in the Matrigel resulted in clear capillary-like formations with an increase of hemoglobin content in the plug. This is the first report showing that apelin is an angiogenic factor in retinal endothelial cells

High-intensity Interval training enhances mobilization/functionality of endothelial progenitor cells and depressed shedding of vascular endothelial cells undergoing hypoxia.

Science.gov (United States)

Tsai, Hsing-Hua; Lin, Chin-Pu; Lin, Yi-Hui; Hsu, Chih-Chin; Wang, Jong-Shyan

2016-12-01

Exercise training improves endothelium-dependent vasodilation, whereas hypoxic stress causes vascular endothelial dysfunction. Monocyte-derived endothelial progenitor cells (Mon-EPCs) contribute to vascular repair process by differentiating into endothelial cells. This study investigates how high-intensity interval (HIT) and moderate-intensity continuous (MCT) exercise training affect circulating Mon-EPC levels and EPC functionality under hypoxic condition. Sixty healthy sedentary males were randomized to engage in either HIT (3-min intervals at 40 and 80 % VO 2max for five repetitions, n = 20) or MCT (sustained 60 % VO 2max , n = 20) for 30 min/day, 5 days/week for 6 weeks, or to a control group (CTL) that did not received exercise intervention (n = 20). Mon-EPC characteristics and EPC functionality under hypoxic exercise (HE, 100 W under 12 % O 2 ) were determined before and after HIT, MCT, and CTL. The results demonstrated that after the intervention, the HIT group exhibited larger improvements in VO 2peak , estimated peak cardiac output (Q C ), and estimated peak perfusions of frontal cerebral lobe (Q FC ) and vastus lateralis (Q VL ) than the MCT group. Furthermore, HIT (a) increased circulating CD14 ++ /CD16 - /CD34 + /KDR + (Mon-1 EPC) and CD14 ++ /CD16 + /CD34 + /KDR + (Mon-2 EPC) cell counts, (b) promoted the migration and tube formation of EPCs, (c) diminished the shedding of endothelial (CD34 - /KDR + /phosphatidylserine + ) cells, and (d) elevated plasma nitrite plus nitrate, stromal cell-derived factor-1, matrix metalloproteinase-9, and vascular endothelial growth factor-A concentrations at rest or following HE, compared to those of MCT. In addition, Mon-1 and -2 EPC counts were directly related to VO 2peak and estimated peak Q C , Q FC , and Q VL . HIT is superior to MCT for improving hemodynamic adaptation and Mon-EPC production. Moreover, HIT effectively enhances EPC functionality and suppresses endothelial injury undergoing hypoxia.

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

Science.gov (United States)

Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

2004-12-01

The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Enhanced adhesion of early endothelial progenitor cells to radiation-induced senescence-like vascular endothelial cells in vitro

International Nuclear Information System (INIS)

Sermsathanasawadi, N.; Inoue, Yoshinori; Iwai, Takehisa; Ishii, Hideto; Yoshida, Masayuki; Igarashi, Kaori; Miura, Masahiko

2009-01-01

The effects of ionizing radiation (IR) on tumor neovascularization are still unclear. We previously reported that vascular endothelial cells (ECs) expressing the IR-induced senescence-like (IRSL) phenotype exhibit a significant decrease in angiogenic activity in vitro. In this study, we examined the effects of the IRSL phenotype on adhesion to early endothelial progenitor cells (early EPCs). Adhesion of human peripheral blood-derived early EPCs to human umbilical vein endothelial cells (HUVECs) expressing the IRSL phenotype was evaluated by an adhesion assay under static conditions. It was revealed that the IRSL HUVECs supported significantly more adhesion of early EPCs than normal HUVECs. Expressions of ICAM-1, VCAM-1 and E-selectin were up-regulated in IRSL HUVECs. Pre-treatment of IRSL HUVECs with adhesion-blocking monoclonal antibodies against E-selectin and VCAM-1 significantly reduced early EPC adhesion to IRSL HUVECs, suggesting a potential role for the E-selectin and VCAM-1 in the adhesion between IRSL ECs and early EPCs. Therefore, the IRSL phenotype expressed in ECs may enhance neovascularization via increased homing of early EPCs. Our findings are first to implicate the complex effects of this phenotype on tumor neovascularization following irradiation. (author)

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

OpenAIRE

Yongliang Yang; Nima Jamilpour; Baoyin Yao; Zachary S. Dean; Reza Riahi; Pak Kin Wong

2016-01-01

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via pe...

Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

Science.gov (United States)

Fearnley, Gareth W; Smith, Gina A; Odell, Adam F; Latham, Antony M; Wheatcroft, Stephen B; Harrison, Michael A; Tomlinson, Darren C; Ponnambalam, Sreenivasan

2014-01-01

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function. © 2014 Elsevier Inc. All rights reserved.

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment

International Nuclear Information System (INIS)

Gery, Bernard; Little, John B.; Coppey, Jacques

1996-01-01

Purpose: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. Methods and Materials: The cell system consists of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. Results: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 10 5 fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. Conclusions: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumors fragments

Endothelial cell capture of heparin-binding growth factors under flow.

Directory of Open Access Journals (Sweden)

Bing Zhao

2010-10-01

Full Text Available Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2 was used for both the experiments and simulations. Our computational model was composed of three parts: (1 media flow equations, (2 mass transport equations and (3 cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that

Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture

International Nuclear Information System (INIS)

Sadlonova, Andrea; Novak, Zdenek; Johnson, Martin R; Bowe, Damon B; Gault, Sandra R; Page, Grier P; Thottassery, Jaideep V; Welch, Danny R; Frost, Andra R

2005-01-01

Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed. NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR. In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation. Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF

Endothelial cell chimerism associated with graft rejection after human lung transplantation.

OpenAIRE

Ratajczak , Philippe; Murata , Hideyuki; Meignin , Véronique; Groussard , Odile; Fournier , Michel; Socié , Gérard; Mal , Hervé; Janin , Anne

2008-01-01

International audience; Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD3...

Radiation-induced chromosome aberrations and cell killing in normal human fibroblasts and ataxia telangiectasia fibroblasts

International Nuclear Information System (INIS)

Kawata, T.; Saito, M.; Uno, T.; Ito, H.; Shigematsu, N.

2003-01-01

Full text: When cells are held in a non-dividing state (G0) after irradiation, an enhanced survival can be observed compared to that of immediate plating. A change of survival depending on post irradiation condition is known to be repair of potentially lethal damage (RPLD). The effects of confluent holding recovery (24-h incubation following irradiation) on chromosome aberrations in normal human fibroblasts (AG1522) and ataxia telangiectasia fibroblasts (GM02052C) were examined. A chemical-induced premature chromosome condensation (PCC) technique with fluorescent in situ hybridization (FISH) was applied to study chromosome aberrations in G2 and M-phase. Results from cell survival showed that the capacity for potentially lethal damage repair was normal in AG1522 cells but very little in GM02052C cells. The frequency of chromosome aberrations in AG1522 cells decreased when cells were allowed to repair for 24-h. Especially complex type exchanges were found to decrease markedly at high doses (4Gy and 6Gy). However, the frequency of chromosome aberrations including complex type exchanges showed little decrease in GM02052C cells. Confluent holding can effectively reduce chromosome aberrations, especially complex type exchanges in normal cells

Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture

Directory of Open Access Journals (Sweden)

Ruth Olmer

2018-05-01

Full Text Available Summary: Endothelial cells (ECs are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability. : In this article, U. Martin and colleagues show the generation of hiPSC endothelial cells in scalable cultures in up to 100 mL culture volume. The generated ECs show in vitro proliferation capacity and a high degree of chromosomal stability after in vitro expansion. The established protocol allows to generate hiPSC-derived ECs in relevant numbers for regenerative approaches. Keywords: hiPSC differentiation, endothelial cells, scalable culture

Nanofiber density determines endothelial cell behavior on hydrogel matrix

Energy Technology Data Exchange (ETDEWEB)

Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

2013-12-01

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

Nanofiber density determines endothelial cell behavior on hydrogel matrix

International Nuclear Information System (INIS)

Berti, Fernanda V.; Rambo, Carlos R.; Dias, Paulo F.; Porto, Luismar M.

2013-01-01

When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

Science.gov (United States)

Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

2016-11-15

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal lymphangioma.

Science.gov (United States)

Way, D; Hendrix, M; Witte, M; Witte, C; Nagle, R; Davis, J

1987-09-01

An endothelial cell line derived from a massive recurrent chyle-containing retroperitoneal lymphangioma was isolated in monolayer culture. Scanning and transmission electron microscopy and immunohistochemistry confirmed a close resemblance to blood vascular endothelium with typical cobblestone morphology, positive immunofluorescence staining for endothelial marker Factor VIII-associated antigen and fibronectin, and prominent Weibel-Palade bodies. The endothelial cells also exhibited other ultrastructural features characteristic of lymphatic endothelium, including sparse microvillous surface projections, overlapping intercellular junctions, and abundant intermediate filaments. This endothelial cell line represents a new source of proliferating lymphatic endothelium for future study, including structural and functional comparison to blood vascular endothelium.

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Lifescience Database Archive (English)

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Radiation response characteristics of human cell in vitro

International Nuclear Information System (INIS)

Hall, E.J.

1987-01-01

Improvements in tissue culture techniques and growth media have made it possible to culture a range of cells of human origin, both normal and malignant. The most recent addition to the list are endothelial cells. Interesting results have been obtained, some of which may have implications in Radiation Therapy. (i) Repair of Potentially Lethal Damage (PLDR) has been observed in all cell lines investigated; cells of normal origin repair PLD at least as well as malignant cells, which makes clinical trials of PLDR inhibitors of doubtful usefulness. (ii) PLD in fibroblasts of human origin appears to have a component that is repaired rapidly, in a matter of minutes, as well as a slower component that takes hours to repair. (iii) Sublethal damage repair, manifest by a dose-rate effect, has also been observed in all human cell lines tested. Cells of normal tissue origin, including fibroblasts and endothelial cells, exhibit a dose-rate effect that is intermediate between that for cells from traditionally resistant tumors (melanoma and osteosarcoma) and cells from more sensitive tumors (neuroblastoma and breast). (iv) Fibroblasts from patients with Ataxia Telangectasia (AT) are much more sensitive to x-rays, with a D/sub o/ about half that for normal human fibroblasts. Nevertheless repair of both PLD and SLD can be demonstrated in these cells

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Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

DEFF Research Database (Denmark)

Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

2017-01-01

Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

DEFF Research Database (Denmark)

Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje

2012-01-01

with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape (‘rounding up’). Increased expression of apoptotic markers indicated...

Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation

Science.gov (United States)

Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.

1983-11-01

Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

International Nuclear Information System (INIS)

Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

1985-01-01

Incubation of [ 35 S]heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of 35 S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of [ 35 S]heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular [ 35 S]heparin proteoglycan after 24 hours and the appearance of free [ 35 S]sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading [ 35 S]heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule

CD117 expression in fibroblasts-like stromal cells indicates unfavorable clinical outcomes in ovarian carcinoma patients.

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Ruixia Huang

Full Text Available The stem cell factor (SCF receptor CD117 (c-kit, is widely used for identification of hematopoietic stem cells and cancer stem cells. Moreover, CD117 expression in carcinoma cells indicates a poor prognosis in a variety of cancers. However the potential expression in tumor microenvironment and the biological and clinical impact are currently not reported. The expression of CD117 was immunohistochemically evaluated in a serial of 242 epithelial ovarian cancer (EOC cases. Thirty-eight out of 242 cases were CD117 positive in fibroblast-like stromal cells and 22 cases were positive in EOC cells. Four cases were both positive in fibroblast-like stromal cells and EOC cells for CD117. CD117 expression in fibroblast-like stromal cells in ovarian carcinoma was closely linked to advanced FIGO stage, poor differentiation grade and histological subtype (p<0.05, and it was significantly associated with poor overall survival (OS and progression free survival (PFS (Kaplan-Meier analysis; p<0.05, log-rank test. CD117 expression in ovarian carcinoma cells was not associated with these clinicopathological variables. The CD117 positive fibroblast-like stromal cells were all positive for mesenchymal stem/stromal cell (MSC marker CD73 but negative for fibroblast markers fibroblast activation protein (FAP and α smooth muscle actin (α-SMA, indicating that the CD117+/CD73+ fibroblast-like stromal cells are a subtype of mesenchymal stem cells in tumor stroma, although further characterization of these cells are needed. It is concluded herewith that the presence of CD117+/CD73+ fibroblast-like stromal cells in ovarian carcinoma is an unfavorable clinical outcome indication.

CD13 is a novel mediator of monocytic/endothelial cell adhesion

DEFF Research Database (Denmark)

Mina-Osorio, Paola; Winnicka, Beata; O'Conor, Catherine

2008-01-01

During inflammation, cell surface adhesion molecules guide the adhesion and migration of circulating leukocytes across the endothelial cells lining the blood vessels to access the site of injury. The transmembrane molecule CD13 is expressed on monocytes and endothelial cells and has been shown...... to mediate homotypic cell adhesion, which may imply a role for CD13 in inflammatory monocyte trafficking. Here, we show that ligation and clustering of CD13 by mAb or viral ligands potently induce myeloid cell/endothelial adhesion in a signal transduction-dependent manner involving monocytic cytoskeletal...... rearrangement and filopodia formation. Treatment with soluble recombinant (r)CD13 blocks this CD13-dependent adhesion, and CD13 molecules from monocytic and endothelial cells are present in the same immunocomplex, suggesting a direct participation of CD13 in the adhesive interaction. This concept...

Recovery of Corneal Endothelial Cells from Periphery after Injury.

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Sang Ouk Choi

Full Text Available Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.To investigate the recovery process of corneal endothelial cells (CECs from corneal endothelial injury.Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group. Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.

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Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

International Nuclear Information System (INIS)

Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

2010-01-01

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

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Origin of Matrix-Producing Cells That Contribute to Aortic Fibrosis in Hypertension.

Science.gov (United States)

Wu, Jing; Montaniel, Kim Ramil C; Saleh, Mohamed A; Xiao, Liang; Chen, Wei; Owens, Gary K; Humphrey, Jay D; Majesky, Mark W; Paik, David T; Hatzopoulos, Antonis K; Madhur, Meena S; Harrison, David G

2016-02-01

Various hypertensive stimuli lead to exuberant adventitial collagen deposition in large arteries, exacerbating blood pressure elevation and end-organ damage. Collagen production is generally attributed to resident fibroblasts; however, other cells, including resident and bone marrow-derived stem cell antigen positive (Sca-1(+)) cells and endothelial and vascular smooth muscle cells, can produce collagen and contribute to vascular stiffening. Using flow cytometry and immunofluorescence, we found that adventitial Sca-1(+) progenitor cells begin to produce collagen and acquire a fibroblast-like phenotype in hypertension. We also found that bone marrow-derived cells represent more than half of the matrix-producing cells in hypertension, and that one-third of these are Sca-1(+). Cell sorting and lineage-tracing studies showed that cells of endothelial origin contribute to no more than one fourth of adventitial collagen I(+) cells, whereas those of vascular smooth muscle lineage do not contribute. Our findings indicate that Sca-1(+) progenitor cells and bone marrow-derived infiltrating fibrocytes are major sources of arterial fibrosis in hypertension. Endothelial to mesenchymal transition likely also contributes, albeit to a lesser extent and pre-existing resident fibroblasts represent a minority of aortic collagen-producing cells in hypertension. This study shows that vascular stiffening represents a complex process involving recruitment and transformation of multiple cells types that ultimately elaborate adventitial extracellular matrix. © 2015 American Heart Association, Inc.

Endothelial and circulating progenitor cells in hematological diseases and allogeneic hematopoietic stem cell transplantation.

Science.gov (United States)

Ruggeri, Annalisa; Paviglianiti, Annalisa; Volt, Fernanda; Kenzey, Chantal; Rafii, Hanadi; Rocha, Vanderson; Gluckman, Eliane

2017-10-12

Circulating endothelial cells (CECs), originated form endothelial progenitors (EPCs) are mature cells which are not associated with vessel walls, and that are detached from the endothelium. Normally, they are present in insignificant amounts in the peripheral blood of healthy individuals. On the other hand, elevated CECs and EPCs levels have been reported in the peripheral blood of patients with different types of cancers and some other diseases. Consequently, CECs and EPCs represent a potential biomarker in several clinical conditions involving endothelial turnover and remodeling, such as hematological diseases. These cells may be involved in disease progression and the neoplastic angiogenesis process. Moreover, CESs and EPCs are probably involved in endothelial damage that is a marker of several complications following allogeneic hematopoietic stem cell transplantation. This review aims to provide an overview on the characterization of CECs and EPCs, describe isolation methods and to identify the potential role of these cells in hematological diseases and hematopoietic stem cell transplantation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The adaptor CRADD/RAIDD controls activation of endothelial cells by proinflammatory stimuli.

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Qiao, Huan; Liu, Yan; Veach, Ruth A; Wylezinski, Lukasz; Hawiger, Jacek

2014-08-08

A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists. © 2014 by The American Society for Biochemistry and Molecular Biology

Tumor necrosis factor-α enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

International Nuclear Information System (INIS)

Song, Kai; Zhu, Fei; Zhang, Han-zhong; Shang, Zheng-jun

2012-01-01

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-α (TNF-α) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-α-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer–endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-α could enhance cancer–endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer–endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: ► Spontaneous oral cancer–endothelial cell fusion. ► TNF-α enhanced cell fusions. ► VCAM-1/VLA-4 expressed in oral cancer. ► TNF-α increased expression of VCAM-1 on endothelial cells. ► VCAM-1/VLA-4 mediated TNF-α-enhanced cell fusions.

Tumor necrosis factor-{alpha} enhanced fusions between oral squamous cell carcinoma cells and endothelial cells via VCAM-1/VLA-4 pathway

Energy Technology Data Exchange (ETDEWEB)

Song, Kai; Zhu, Fei; Zhang, Han-zhong [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); Shang, Zheng-jun, E-mail: shangzhengjun@hotmail.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST), Key Laboratory for Oral Biomedicine Ministry of Education, Wuhan University, Wuhan (China); First Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, Wuhan (China)

2012-08-15

Fusion between cancer cells and host cells, including endothelial cells, may strongly modulate the biological behavior of tumors. However, no one is sure about the driving factors and underlying mechanism involved in such fusion. We hypothesized in this study that inflammation, one of the main characteristics in tumor microenvironment, serves as a prominent catalyst for fusion events. Our results showed that oral cancer cells can fuse spontaneously with endothelial cells in co-culture and inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}) increased fusion of human umbilical vein endothelium cells and oral cancer cells by up to 3-fold in vitro. Additionally, human oral squamous cell carcinoma cell lines and 35 out of 50 (70%) oral squamous carcinoma specimens express VLA-4, an integrin, previously implicated in fusions between human peripheral blood CD34-positive cells and murine cardiomyocytes. Expression of VCAM-1, a ligand for VLA-4, was evident on vascular endothelium of oral squamous cell carcinoma. Moreover, immunocytochemistry and flow cytometry analysis revealed that expression of VCAM-1 increased obviously in TNF-{alpha}-stimulated endothelial cells. Anti-VLA-4 or anti-VCAM-1 treatment can decrease significantly cancer-endothelial adhesion and block such fusion. Collectively, our results suggested that TNF-{alpha} could enhance cancer-endothelial cell adhesion and fusion through VCAM-1/VLA-4 pathway. This study provides insights into regulatory mechanism of cancer-endothelial cell fusion, and has important implications for the development of novel therapeutic strategies for prevention of metastasis. -- Highlights: Black-Right-Pointing-Pointer Spontaneous oral cancer-endothelial cell fusion. Black-Right-Pointing-Pointer TNF-{alpha} enhanced cell fusions. Black-Right-Pointing-Pointer VCAM-1/VLA-4 expressed in oral cancer. Black-Right-Pointing-Pointer TNF-{alpha} increased expression of VCAM-1 on endothelial cells. Black

Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells.

Science.gov (United States)

Khor, Hwei Ling; Kuan, Yujun; Kukula, Hildegard; Tamada, Kaoru; Knoll, Wolfgang; Moeller, Martin; Hutmacher, Dietmar W

2007-05-01

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.

[Circulating endothelial cells: biomarkers for monitoring activity of antiangiogenic therapy].

Science.gov (United States)

Farace, Françoise; Bidart, Jean-Michel

2007-07-01

Tumor vessel formation is largely dependent on the recruitment of endothelial cells. Rare in healthy individuals, circulating endothelial cells (CEC) are shed from vessel walls and enter the circulation reflecting endothelial damage or dysfunction. Increased numbers of CEC have been documented in different types of cancer. Recent studies have suggested the role for CEC in tumor angiogenesis, but whose presence could also reflect normal endothelium perturbation in cancer. Originating from the bone marrow rather than from vessel walls, endothelial progenitor cells (EPC) are mobilized following tissue ischemia and may be recruited to complement local angiogenesis supplied by existing endothelium. Recently, studies in mouse models suggest that the circulating fraction of endothelial progenitors (CEP) is involved in tumor angiogenesis but their contribution is less clear in humans. The detection of CEC and CEP is difficult and impeded by the rarity of these cells. They may have important clinical implication as novel biomarkers susceptible to predict more efficiently and rapidly the therapeutic response to anti-angiogenic treatments. However, a methodological consensus would be necessary in order to correctly evaluate the clinical interest of CEC and CEP in patients.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

Science.gov (United States)

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

Wound-healing potential of human umbilical cord blood-derived mesenchymal stromal cells in vitro--a pilot study.

Science.gov (United States)

You, Hi-Jin; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

2015-11-01

Our previous studies demonstrated that human bone marrow-derived mesenchymal stromal cells have great potential for wound healing. However, it is difficult to clinically utilize cultured stem cells. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialized for cartilage repair as a first cell therapy product that uses allogeneic stem cells. Should hUCB-MSCs have a superior effect on wound healing as compared with fibroblasts, which are the main cell source in current cell therapy products for wound healing, they may possibly replace fibroblasts. The purpose of this in vitro study was to compare the wound-healing activity of hUCB-MSCs with that of fibroblasts. This study was particularly designed to compare the effect of hUCB-MSCs on diabetic wound healing with those of allogeneic and autologous fibroblasts. Healthy (n = 5) and diabetic (n = 5) fibroblasts were used as the representatives of allogeneic and autologous fibroblasts for diabetic patients in the control group. Human UCB-MSCs (n = 5) were used in the experimental group. Cell proliferation, collagen synthesis and growth factor (basic fibroblast growth factor, vascular endothelial growth factor and transforming growth factor-β) production were compared among the three cell groups. Human UCB-MSCs produced significantly higher amounts of vascular endothelial growth factor and basic fibroblast growth factor when compared with both fibroblast groups. Human UCB-MSCs were superior to diabetic fibroblasts but not to healthy fibroblasts in collagen synthesis. There were no significant differences in cell proliferation and transforming growth factor-β production. Human UCB-MSCs may have greater capacity for diabetic wound healing than allogeneic or autologous fibroblasts, especially in angiogenesis. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Radiosensitization of human endothelial cells by IL-24

International Nuclear Information System (INIS)

Meyn, R.E.

2003-01-01

Radiation therapy remains an important cancer treatment modality but despite improvements in dose delivery many patients still fail at their primary tumor site. Therefore, new strategies designed to improve local control are needed. Protocols combining radiation with anti-angiogenic agents might be of particular advantage based on their documented low toxicity. In this regard, we have been conducting preclinical investigations of a novel cytokine, mda7/IL-24. Our collaborators have shown that mda7/IL-24 protein targets the endothelial cells of the tumor microvascular system and has potent anti-angiogenic properties in both in vitro and in vivo assays. Recently, we have demonstrated that recombinant mda7/IL-24 protein radiosensitizes human endothelial cells in vitro. Specifically, 10 ng/ml of recombinant human IL-24 protein for 12 hrs reduced the survival at 2 Gy for human umbilical vein endothelial cells (HUVECs) from 0.33 to 0.12. We are also working on understanding the molecular basis for this radiosensitizing effect. Preliminary data suggest a model whereby mda7/IL-24 engages a specific receptor on the surface of endothelial cells and initiates a signal transduction pathway that modulates the cell's propensity for radiation-induced apoptosis and capacity for repairing radiation-induced DNA double strand breaks. Mechanistic insight gained from these studies may have implications for the actions of other anti-angiogenic agents and may generally explain the regulation of radiosensitivity imparted by growth factors and cytokines

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

Directory of Open Access Journals (Sweden)

Cristina Espinosa-Díez

2018-04-01

Full Text Available Glutathione (GSH biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL, which is composed of the catalytic (GCLc and the modulatory (GCLm subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice. In murine lung endothelial cells (MLEC derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177 and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+ male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+ mice. We observed that obstructed kidneys from Gclc(e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Keywords: Glutamate-cysteine ligase, ROS, Glutathione, Endothelial dysfunction, Kidney Fibrosis

Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model.

Directory of Open Access Journals (Sweden)

Rie Kawabe-Yako

Full Text Available BACKGROUND: Cilostazol(CLZ has been used as a vasodilating anti-platelet drug clinically and demonstrated to inhibit proliferation of smooth muscle cells and effect on endothelial cells. However, the effect of CLZ on re-endothelialization including bone marrow (BM-derived endothelial progenitor cell (EPC contribution is unclear. We have investigated the hypothesis that CLZ might accelerate re-endothelialization with EPCs. METHODOLOGY/PRINCIPAL FINDINGS: Balloon carotid denudation was performed in male Sprague-Dawley rats. CLZ group was given CLZ mixed feed from 2 weeks before carotid injury. Control group was fed normal diet. CLZ accelerated re-endothelialization at 2 weeks after surgery and resulted in a significant reduction of neointima formation 4 weeks after surgery compared with that in control group. CLZ also increased the number of circulating EPCs throughout the time course. We examined the contribution of BM-derived EPCs to re-endothelialization by BM transplantation from Tie2/lacZ mice to nude rats. The number of Tie2-regulated X-gal positive cells on injured arterial luminal surface was increased at 2 weeks after surgery in CLZ group compared with that in control group. In vitro, CLZ enhanced proliferation, adhesion and migration activity, and differentiation with mRNA upregulation of adhesion molecule integrin αvβ3, chemokine receptor CXCR4 and growth factor VEGF assessed by real-time RT-PCR in rat BM-derived cultured EPCs. In addition, CLZ markedly increased the expression of SDF-1α that is a ligand of CXCR4 receptor in EPCs, in the media following vascular injury. CONCLUSIONS/SIGNIFICANCE: CLZ promotes EPC mobilization from BM and EPC recruitment to sites of arterial injury, and thereby inhibited neointima formation with acceleration of re-endothelialization with EPCs as well as pre-existing endothelial cells in a rat carotid balloon injury model. CLZ could be not only an anti-platelet agent but also a promising tool for

Hyaluronan protection of corneal endothelial cells against extracellular histones after phacoemulsification.

Science.gov (United States)

Kawano, Hiroki; Sakamoto, Taiji; Ito, Takashi; Miyata, Kazunori; Hashiguchi, Teruto; Maruyama, Ikuro

2014-11-01

To determine the effect of histones on corneal endothelial cells generated during cataract surgery. Kagoshima University Hospital, Kagoshima, Japan. Experimental study. Standard phacoemulsification was performed on enucleated pig eyes. Histones in the anterior segment of the eye were determined by immunohistochemistry. Cultured human corneal endothelial cells were exposed to histones for 18 hours, and cell viability was determined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt assay. The concentration of interleukin-6 (IL-6) in the culture medium of human corneal endothelial cells was measured using enzyme-linked immunosorbent assay. The effects of signal inhibitors U0126, SB203580, and SP600125 were evaluated. The protective effect of hyaluronan against histones was evaluated in human corneal endothelial cells with and without hyaluronan. Cellular debris containing histones was observed in the anterior chamber of pig eyes after phacoemulsification. Exposure of human corneal endothelial cells to 50 μg/mL of histones or more led to cytotoxic effects. The IL-6 concentration was significantly increased dose dependently after exposure of human corneal endothelial cells to histones (Phistone-induced IL-6 production was significantly decreased by extracellular signal-regulated kinases 1/2 and p-38 mitogen-activated protein kinase inhibitors (Phistones caused formation of histone aggregates, decreased the cytotoxic effects of the histones, and blocked the increase in IL-6 (PHistones were released extracellularly during phacoemulsification and exposure of human corneal endothelial cells to histones increased the IL-6 secretion. The intraoperative use of hyaluronan may decrease the cytotoxic effects of histones released during cataract surgery. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

Science.gov (United States)

Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

2013-01-01

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

The targeting expression of the vascular endothelial growth factor gene in endothelial cells regulated by HRE.ppET-1.

Science.gov (United States)

Zheng, Xiangrong; Zhang, Shangshang; Yang, Yujia; Wang, Xia; Zhong, Le; Yu, Xiaohe

2008-11-01

The success of gene therapy depends largely on the efficacy of gene delivery vector systems that can deliver genes to target organs or cells selectively and efficiently with minimal toxicity. Here, we show that by using the HRE.ppET-1 regulatory element, we were able to restrict expression of the transgene of vascular endothelial growth factor (VEGF) to endothelial cells exclusively in hypoxic conditions. Eukaryotic expression vectors such as pEGFP-HRE.ppET-1, pcDNA3.1-VEGF+Pa, pcDNA3.1-ppET-1+ EGF+Pa, and pcDNA3.1-HRE.ppET-1+VEGF+Pa were constructed by using a series of nuclear molecule handling methods like PCR, enzyme digestion. The recombinant vectors were transfected into HUVEC cells and HL7702 cells by the lipofectin method. GFP expression was observed with a fluorescence microscope to validate the specificity of expression in endothelial cells under the regulation of HRE.ppET-1 element. Cobalt chloride (final concentration 100 mumol/L) was added to the medium to mimic hypoxia in vitro. After transfection of vectors, the expression of VEGF mRNA was detected by RT-PCR, and the expression of VEGF was detected by Western blotting and ELISA methods under normoxia and hypoxia, respectively. The cell proliferation rate was detected by the MTT test. The expression of GFP revealed that the exterior gene was transcripted effectively in endothelial cells regulated by the HRE.ppET-1 element, while the expression of GFP was very weak in nonendothelial cells. The results of RT-PCR, Western blotting and ELISA showed that VEGF gene expression in the pcDNA3.1-HRE.ppET-1+VEGF+Pa group and in the pcDNA3.1-ppET-1+VEGF+Pa group was higher in hypoxia than it was in normoxia (PHRE.ppET-1 element was expressed specifically in endothelial cells, and can increase the expression of VEGF in hypoxia and stimulate proliferation of endothelial cells. Taking advantage of these facts could greatly improve the efficiency of gene therapy. The vector would be valuable for various gene transfer

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

Science.gov (United States)

Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian Gabriel

2017-01-01

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro

International Nuclear Information System (INIS)

Rodemann, H.P.; Bayreuther, K.; Francz, P.I.; Dittmann, K.; Albiez, M.

1989-01-01

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern

Cytomegalovirus-Induced Effector T Cells Cause Endothelial Cell Damage

NARCIS (Netherlands)

van de Berg, Pablo J. E. J.; Yong, Si-La; Remmerswaal, Ester B. M.; van Lier, René A. W.; ten Berge, Ineke J. M.

2012-01-01

Human cytomegalovirus (CMV) infection has been linked to inflammatory diseases that involve vascular endothelial cell damage, but definitive proof for a direct cytopathic effect of CMV in these diseases is lacking. CMV infection is associated with a strong increase in both CD4(+) and CD8(+) T cells

Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts

International Nuclear Information System (INIS)

Samoszuk, Michael; Tan, Jenny; Chorn, Guillaume

2005-01-01

Accumulating evidence suggests that fibroblasts play a pivotal role in promoting the growth of breast cancer cells. The objective of the present study was to characterize and validate an in vitro model of the interaction between small numbers of human breast cancer cells and human fibroblasts. We measured the clonogenic growth of small numbers of human breast cancer cells co-cultured in direct contact with serum-activated, normal human fibroblasts. Using DNA microarrays, we also characterized the gene expression profile of the serum-activated fibroblasts. In order to validate the in vivo relevance of our experiments, we then analyzed clinical samples of metastatic breast cancer for the presence of myofibroblasts expressing α-smooth muscle actin. Clonogenic growth of human breast cancer cells obtained directly from in situ and invasive tumors was dramatically and consistently enhanced when the tumor cells were co-cultured in direct contact with serum-activated fibroblasts. This effect was abolished when the cells were co-cultured in transwells separated by permeable inserts. The fibroblasts in our experimental model exhibited a gene expression signature characteristic of 'serum response' (i.e. myofibroblasts). Immunostaining of human samples of metastatic breast cancer tissue confirmed that myofibroblasts are in direct contact with breast cancer cells. Serum-activated fibroblasts promote the clonogenic growth of human breast cancer cells in vitro through a mechanism that involves direct physical contact between the cells. This model shares many important molecular and phenotypic similarities with the fibroblasts that are naturally found in breast cancers

Endothelial cell adhesion to ion implanted polymers

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Y; Kusakabe, M [SONY Corp., Tokyo (Japan); Lee, J S; Kaibara, M; Iwaki, M; Sasabe, H [RIKEN (Inst. of Physical and Chemical Research), Saitama (Japan)

1992-03-01

The biocompatibility of ion implanted polymers has been studied by means of adhesion measurements of bovine aorta endothelial cells in vitro. The specimens used were polystyrene (PS) and segmented polyurethane (SPU). Na{sup +}, N{sub 2}{sup +}, O{sub 2}{sup +} and Kr{sup +} ion implantations were performed at an energy of 150 keV with fluences ranging from 1x10{sup 15} to 3x10{sup 17} ions/cm{sup 2} at room temperature. The chemical and physical structures of ion-implanted polymers have been investigated in order to analyze their tissue compatibility such as improvement of endothelial cell adhesion. The ion implanted SPU have been found to exhibit remarkably higher adhesion and spreading of endothelial cells than unimplanted specimens. By contrast, ion implanted PS demonstrated a little improvement of adhesion of cells in this assay. Results of FT-IR-ATR showed that ion implantation broke the original chemical bond to form new radicals such as OH, ....C=O, SiH and condensed rings. The results of Raman spectroscopy showed that ion implantation always produced a peak near 1500 cm{sup -1}, which indicated that these ion implanted PS and SPU had the same carbon structure. This structure is considered to bring the dramatic increase in the extent of cell adhesion and spreading to these ion implanted PS and SPU. (orig.).

Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts

Directory of Open Access Journals (Sweden)

Maucksch C

2012-01-01

Full Text Available Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP-expressing astrocytes. This study represents a novel virus-free approach for direct reprogramming of human fibroblasts to a neural precursor fate.

Endothelial glycocalyx on brain endothelial cells is lost in experimental cerebral malaria

DEFF Research Database (Denmark)

Hempel, Casper; Hyttel, Poul; Kurtzhals, Jørgen Al

2014-01-01

We hypothesized that the glycocalyx, which is important for endothelial integrity, is lost in severe malaria. C57BL/6 mice were infected with Plasmodium berghei ANKA, resulting in cerebral malaria, or P. chabaudi AS, resulting in uncomplicated malaria. We visualized the glycocalyx with transmission...... electron microscopy and measured circulating glycosaminoglycans by dot blot and ELISA. The glycocalyx was degraded in brain vasculature in cerebral and to a lesser degree uncomplicated malaria. It was affected on both intact and apoptotic endothelial cells. Circulating glycosaminoglycan levels suggested...

A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

Science.gov (United States)

Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

2017-11-01

Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P livers correlated with a significant decrease in liver fibrosis ( P livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American

Tolerogenic properties of lymphatic endothelial cells are controlled by the lymph node microenvironment.

Directory of Open Access Journals (Sweden)

Jarish N Cohen

Full Text Available Peripheral self-tolerance eliminates lymphocytes specific for tissue-specific antigens not encountered in the thymus. Recently, we demonstrated that lymphatic endothelial cells in mice directly express peripheral tissue antigens, including tyrosinase, and induce deletion of specific CD8 T cells via Programmed Death Ligand-1 (PD-L1. Here, we demonstrate that high-level expression of peripheral tissue antigens and PD-L1 is confined to lymphatic endothelial cells in lymph nodes, as opposed to tissue (diaphragm and colon lymphatics. Lymphatic endothelial cells in the lymph node medullary sinus express the highest levels of peripheral tissue antigens and PD-L1, and are the only subpopulation that expresses tyrosinase epitope. The representation of lymphatic endothelial cells in the medullary sinus expressing high-level PD-L1, which is necessary for normal CD8 T cell deletion kinetics, is controlled by lymphotoxin-β receptor signaling and B cells. Lymphatic endothelial cells from neonatal mice do not express high-level PD-L1 or present tyrosinase epitope. This work uncovers a critical role for the lymph node microenvironment in endowing lymphatic endothelial cells with potent tolerogenic properties.

Coniferyl aldehyde attenuates radiation enteropathy by inhibiting cell death and promoting endothelial cell function.

Science.gov (United States)

Jeong, Ye-Ji; Jung, Myung Gu; Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

2015-01-01

Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.

Comparison of fibroblast cell regeneration in three different concentrations of Wharton’s Jelly mesenchymal stem cells conditioned medium (WJMSCs-CM)

Science.gov (United States)

Untoro, E. G.; Asrianti, D.; Usman, M.; Meidyawati, R.; Margono, A.

2017-08-01

Wharton’s Jelly-derived mesenchymal stem cells (WJMSCs) have gained interest as an alternative source of stem cells for regenerative medicine. Although many studies have characterized Wharton’s Jelly biologically, the effects of different concentrations in a cultured medium have not yet been compared. Damaged fibroblasts, the primary components of irreversible dental pulpitis, irreversibly impair the ability to regenerate and lead to the disruption of extracellular matrix. This study was performed to evaluate the potency of three WJMSCs-CM concentrations in improving serum-starved fibroblasts. Fibroblasts were cultivated in five passages, and divided into four groups. The first group (the control group) consisted of fibroblast cells that had been treated using starvation methods. The other groups (the treatment groups) were treated with various concentration of WJMSCs-CM (50%, 25% and 12.5%). Proliferative ability was evaluated using a cell count method and analyzed with a one-way ANOVA. Cultivation of serum-starved fibroblasts produced significantly higher cell counts in 12.5% WJMSCs-CM compared to the 50% group. It can be concluded that 12.5% WJMSCs-CM is the most efficient concentration for fibroblast proliferation.

Organizational behavior of human umbilical vein endothelial cells

Science.gov (United States)

1982-01-01

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization. PMID:6813338

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

Energy Technology Data Exchange (ETDEWEB)

Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

2014-04-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

International Nuclear Information System (INIS)

Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

2014-01-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM TGF , FCM PDGF ) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM B ). FCM TGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM TGF ≫FCM PDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM TGF >FCM PDGF ) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify

Responses of fibroblasts and glial cells to nanostructured platinum surfaces

Energy Technology Data Exchange (ETDEWEB)

Pennisi, C P; Sevcencu, C; Yoshida, K [Center for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg (Denmark); Dolatshahi-Pirouz, A; Foss, M; Larsen, A Nylandsted; Besenbacher, F [Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus (Denmark); Hansen, J Lundsgaard [Department of Physics and Astronomy, Aarhus University, Aarhus (Denmark); Zachar, V, E-mail: cpennisi@hst.aau.d [Laboratory for Stem Cell Research, Aalborg University (Denmark)

2009-09-23

The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

Improved endothelial cell seeding with cultured cells and fibronectin-coated grafts

International Nuclear Information System (INIS)

Seeger, J.M.; Klingman, N.

1985-01-01

A possible approach to the low seeding efficiency of endothelial cells into prosthetic grafts is to increase the number of cells to be seeded in cell culture and improve seeding efficiency by graft precoating with fibronectin. The effect of cell culture on cell adhesion is unknown, however, and fibronectin also binds fibrin, which may increase the thrombogenicity of the graft luminal surface. To investigate these questions, freshly harvested canine jugular vein endothelial cells from six animals and similar cells harvested from six primary and eight secondary cell cultures were labeled with 111 Indium and seeded into 5 cm, 4 mm PTFE grafts coated with fibronectin, using similar uncoated PTFE grafts as controls. Platelet accumulation and distribution on six similar coated and uncoated grafts placed in canine carotid, external jugular arterial venous shunts for 2 hr were also determined using autogenous 111 Indium-labeled platelets. Significant differences between group means were determined using the paired Student's t test. Results reveal that seeding efficiency is significantly better in all groups of coated grafts compared to uncoated grafts (P less than 0.01). Cells derived from cell culture also had significantly higher seeding efficiencies than freshly harvested cells when seeded into coated grafts (P less than 0.05) and tended to have higher seeding efficiencies than harvested cells when seeded into uncoated grafts (P = 0.53). Fibronectin coating increased mean platelet accumulation on the entire graft luminal surface, but not to a statistically significant degree (P greater than 0.1). Whether this increased seeding efficiency will improve graft endothelialization remains to be investigated

Acrylamide induces accelerated endothelial aging in a human cell model.

Science.gov (United States)

Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

2015-09-01

Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endogenous Vascular Endothelial Growth Factor-A (VEGF-A) Maintains Endothelial Cell Homeostasis by Regulating VEGF Receptor-2 Transcription*

Science.gov (United States)

E, Guangqi; Cao, Ying; Bhattacharya, Santanu; Dutta, Shamit; Wang, Enfeng; Mukhopadhyay, Debabrata

2012-01-01

Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis. PMID:22167188

Oxidative Stress Induces Endothelial Cell Senescence via Downregulation of Sirt6

Directory of Open Access Journals (Sweden)

Rong Liu

2014-01-01

Full Text Available Accumulating evidence has shown that diabetes accelerates aging and endothelial cell senescence is involved in the pathogenesis of diabetic vascular complications, including diabetic retinopathy. Oxidative stress is recognized as a key factor in the induction of endothelial senescence and diabetic retinopathy. However, specific mechanisms involved in oxidative stress-induced endothelial senescence have not been elucidated. We hypothesized that Sirt6, which is a nuclear, chromatin-bound protein critically involved in many pathophysiologic processes such as aging and inflammation, may have a role in oxidative stress-induced vascular cell senescence. Measurement of Sirt6 expression in human endothelial cells revealed that H2O2 treatment significantly reduced Sirt6 protein. The loss of Sirt6 was associated with an induction of a senescence phenotype in endothelial cells, including decreased cell growth, proliferation and angiogenic ability, and increased expression of senescence-associated β-galactosidase activity. Additionally, H2O2 treatment reduced eNOS expression, enhanced p21 expression, and dephosphorylated (activated retinoblastoma (Rb protein. All of these alternations were attenuated by overexpression of Sirt6, while partial knockdown of Sirt6 expression by siRNA mimicked the effect of H2O2. In conclusion, these results suggest that Sirt6 is a critical regulator of endothelial senescence and oxidative stress-induced downregulation of Sirt6 is likely involved in the pathogenesis of diabetic retinopathy.

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

International Nuclear Information System (INIS)

Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

1987-01-01

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51 Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51 Cr release from radiolabeled monolayers

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

Science.gov (United States)

Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

2016-03-03

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

The apelin receptor influences biomechanical and morphological properties of endothelial cells.

Science.gov (United States)

Strohbach, Anne; Pennewitz, Malte; Glaubitz, Michael; Palankar, Raghavendra; Groß, Stefan; Lorenz, Florian; Materzok, Ilka; Rong, Alena; Busch, Mathias C; Felix, Stephan B; Delcea, Mihaela; Busch, Raila

2018-08-01

The adaption of endothelial cells to local flow conditions is a multifunctional process which leads to distinct alterations in cell shape, the subcellular distribution of structural proteins, and cellular function. G-protein-coupled receptors (GPCRs) have been identified to be fundamentally involved in such processes. Recently, we and others have shown that the expression of the endothelial GPCR apelin receptor (APJ) is regulated by fluid flow and that activation of APJ participates in signaling pathways which are related to processes of mechanotransduction. The present study aims to illuminate these findings by further visualization of APJ function. We show that APJ is located to the cellular junctions and might thus be associated with platelet endothelial cell adhesion molecule-1 (PECAM-1) in human umbilical vein endothelial cells (HUVEC). Furthermore, siRNA-mediated silencing of APJ expression influences the shear-induced adaption of HUVEC in terms of cytoskeletal remodeling, cellular elasticity, cellular motility, attachment, and distribution of adhesion complexes. Taken together, our results demonstrate that APJ is crucial for complemented endothelial adaption to local flow conditions. © 2018 Wiley Periodicals, Inc.

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

International Nuclear Information System (INIS)

Rubin, D.B.; Drab, E.A.; Bauer, K.D.

1989-01-01

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

Endothelial cell seeding on crosslinked collagen : Effects of crosslinking on endothelial cell proliferation and functional parameters

NARCIS (Netherlands)

Wissink, MJB; van Luyn, MJA; Dijk, F; Poot, AA; Engbers, GHM; Beugeling, T; van Aken, WG; Feijen, J

Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, such as crosslinked collagen. Commonly used crosslinking agents such as glutaraldehyde and formaldehyde cause, however, cytotoxic reactions and thereby hamper

Endothelial cell oxidative stress and signal transduction

Directory of Open Access Journals (Sweden)

ROCIO FONCEA

2000-01-01

Full Text Available Endothelial dysfunction (ED is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Increased reactive oxygen species (ROS have been implicated as important mechanisms that contribute to ED, and ROS’s may function as intracellular messengers that modulate signaling pathways. Several intracellular signal events stimulated by ROS have been defined, including the identification of two members of the mitogen activated protein kinase family (ERK1/2 and big MAP kinase, BMK1, tyrosine kinases (Src and Syk and different isoenzymes of PKC as redox-sensitive kinases. ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NFkB and others that result in changes in gene expression and modifications in cellular responses. In order to understand the intracellular mechanisms induced by ROS in endothelial cells (EC, we are studying the response of human umbilical cord vein endothelial cells to increased ROS generation by different pro-atherogenic stimuli. Our results show that Homocysteine (Hcy and oxidized LDL (oxLDL enhance the activity and expression of oxidative stress markers, such as NFkB and heme oxygenase 1. These results suggest that these pro-atherogenic stimuli increase oxidative stress in EC, and thus explain the loss of endothelial function associated with the atherogenic process

Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast

Directory of Open Access Journals (Sweden)

Liguo Sun

2016-01-01

Full Text Available Mesenchymal stem cells (MSCs and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15% at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15% at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15% at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

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Jagged gives endothelial tip cells an edge.

Science.gov (United States)

Suchting, Steven; Eichmann, Anne

2009-06-12

Sprouting blood vessels have tip cells that lead and stalk cells that follow. Benedito et al. (2009) now show that competition between endothelial cells for the tip position is regulated by glycosylation of Notch receptors and by the opposing actions of the Notch ligands Jagged1 and Delta-like 4.